Journal of Ethnopharmacology 284 (2022) 114647

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Catharanthus roseus (L.) G. Don: A review of its ethnobotany,

phytochemistry, ethnopharmacology and toxicities
Sunil Kumar a, Bikarma Singh b, c, *, Ramesh Singh d
a
Department of Chemistry, Ma. Kanshiram Government Degree College, Ninowa, (affiliated to Chhatrapati Shahu Ji Maharaj University (CSJM) Kanpur), Farrukhabad,
209602, Uttar Pradesh, India
b
Botanic Garden Division, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow, 226001, Uttar Pradesh, India
c
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, Uttar Pradesh, India
d
Department of Botany, Government Degree College Bahua Dehat, (affiliated to Professor Rajendra Singh (Rajju Bhaiya) University Prayagraj), Fatehpur, 212663, Uttar
Pradesh, India

A R T I C L E I N F O A B S T R A C T

Keywords: Ethnopharmacological relevance: Catharanthus roseus (L.) G. Don is a well known medicinal plant belonging to
Madagascar periwinkle Or periwinkle plant family Apocynaceae that have been traditionally used as medicine since ancient times. C. roseus is a well-
Vinca alkaloids recognized herbal medicine due to its anticancer bisindole alkaloids (vinblastine (111), vincristine (112) and
Traditional medicine
vindesine (121)). In the Ayurvedic system of medicine, different parts of C. roseus are used in folklore herbal
Anticancer activity and quality control
medicine for treatment of many types of cancer, diabetes, stomach disorders, kidney, liver and cardiovascular
Monoterpene indole alkaloids /bisindole
alkaloids diseases.
Analytical methods. pharmacological activity Aim of the study: The main idea behind this communication is to update comprehensively and analyze critically
Toxicology the traditional applications, phytochemistry, pharmacological activities, and toxicity of various extracts and
isolated compounds from C. roseus.
Materials and methods: The presented data covers scientific works on C. roseus published across the world between
1967 and 2021 was searched from various international publishing houses using search engines as well as several
traditional texts like Ayurveda and relevant books. Collected data from different sources was comprehensively
summarized/analyzed for ethnomedicinal uses, phytochemistry, analytical chemistry, biological activities and
toxicity studies of C. roseus.
Results and discussion: C. roseus has a wide range of applications in the traditional system of medicine especially in
cancer and diabetes. During phytochemical investigation, total of 344 compounds including monoterpene indole
alkaloids (MIAs) (110), bisindole alkaloids (35), flavonoids (34), phenolic acids (9) and volatile constituents
(156) have been reported in the various extracts and fractions of different plant parts of C. roseus. The extracts
and isolated compounds of C. roseus have to exhibit many pharmacological activities such as anticancer/cyto­
toxic, antidiabetic, antimicrobial, antioxidant, larvicidal and pupicidal. The comparative toxicity of extracts and
bioactive compounds investigated in dose dependent manner. The investigation of toxicity showed that the both
extracts and isolated compounds are safe to a certain limit beyond that they cause adverse effects.
Conclusion: This review is a comprehensive, critically analyzed summarization of sufficient baseline information
of selected topics in one place undertaken till date on C. roseus for future works and drug discovery. The
phytochemical investigation including biosynthetic pathways showed that the MIAs and bisindole alkaloids are
major and characteristic class of compounds in this plant. The present data confirm that the extracts/fractions
and their isolated alkaloids especially vinblastine (111) and vincristine (112) have a potent anticancer/cytotoxic
and antidiabetic property and there is a need for further study with particular attention to the mechanisms of
anticancer activity. In biosynthesis pathways of alkaloids especially bisindole alkaloids, some enzymes and
rearrangement are unexposed therefore it is required to draw special attention. It also focuses on attracting the
attention of scientific communities about the widespread biological activities of this species for its better utili­
zation prospects in the near future.

* Corresponding author. Botanic Garden Division, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow, 226001, Uttar Pradesh, India.
E-mail address: drbikarma.singh@nbri.res.in (B. Singh).

https://doi.org/10.1016/j.jep.2021.114647
Received 30 April 2021; Received in revised form 7 September 2021; Accepted 14 September 2021
Available online 22 September 2021
0378-8741/© 2021 Elsevier B.V. All rights reserved.
S. Kumar et al.
1. Introduction
Plant as a natural medicine has been used by the people since ancient
times (Singh, 2020). Catharanthus roseus (L.) G.Don belongs to the
dogbane family Apocynaceae is one of the most explored short-lived
medicinal plant species popularly called “Sadabahar” in some Asian
countries such as India, Pakistan and Nepal (Jacobs et al., 2004; Naeem
et al., 2019). This plant species is native to Madagascar. C. roseus is
usually grown as medicinal as well as ornamental plants in parks, gar­
dens, farms and to beautify landscape places (Adekomi, 2020). C. roseus
has an ancient past in Asian and African countries where this plant
species was planted near temples and gardens (Shukla et al., 2010). The
different plant parts of C. roseus are used traditionally in the Indian
system of medicine such as Ayurveda, Siddha, Unani and traditional
Chinese medicine (TCM) (Das and Sharangi, 2017). It is employed for
the treatment of cancer, genitourinary system, kidney, liver, cardio­
vascular diseases (Zhu et al., 2015). The flower is used due to their
fragrance and brain-stimulating odour. The extracts of different parts
and its fractions, and isolated characterized compounds found to have
various pharmacological properties such as antimicrobial, antioxidant,
anthelmintic, antifeedant, antisterility, antidiarrheal and antidiabetic
(Gajalakshmi et al., 2013).
In the last four decades, researchers across the globe showed much
interest in the C. roseus because it contains anticancer bisindole/
monoterpene indole alkaloids (MIAs) and several of these known com­
pounds exhibits very strong and potent pharmacological properties
(Dutta et al., 2005; Mishra and Verma, 2017; Barkat et al., 2017; Kumar
et al., 2018a). The marker compounds vinblastine (111), vincristine
(112) vindesine (121)and vinorelbine (143) are characteristic bisindole
alkaloids that have been permitted for clinical use in the U.S. Food and
Drug Administration for chemotherapy and other medicinal use in the
pharma sectors (Tsuruo et al., 1981; Osterlind et al., 1982; Pennanen
and Huhtikangas, 1990; Kumar et al., 2018b; Al-Quteimat, 2020). These
compounds (111, 112 and 121) are more expensive and have high de­
mand in the global market. Besides, there are several other limited
shoot-specific bioactive compounds present in this plant species which
are found to have medicinal use in pharmaceutical sectors (Yamamoto
et al., 2016). The market prices for compounds 111 and 112 are esti­
mated at $2 million/kg, and $15 million/kg, respectively. The supply of
both compounds 111 and 112 are limited by the availability of the plant
worldwide. The annual market value of compound 111 and 112 are
about $1,000,000–3,500,000 per kg. The costs of velbane and velsar are
made from vinblastine sulfate $ ̃ 2/mg or $ 2 million/kg and are used in
the treatment of many types of cancer like testicular cancer, Hodgkin’s
disease and Kaposi’s sarcoma. Similarly, vincristine sulfate based drugs
(Oncovin and Vincasar PFS) costs ̃$ 15/mg or $ 15 million/kg and are
used for the treatment of leukemia in children (Alam and Sharaf-Eldin,
2016).
The significance of C. roseus in the modern system of medicine has
been recognized after innovative characterization of anticancer bisin­
dole alkaloids such as compounds 111 and 112, which mainly present in
its leaves. The isolation and purification process of both compounds 111
and 112 from leaves are very tedious, time-consuming and costly due to
the presence of very low content of these compounds. Recently, many
advanced and high-throughput separation analytical techniques have
been developed for isolation, purification, identification, characteriza­
tion and quantitation of crude extract prepared from C. roseus (Lin et al.,
2015; Parthasarathy et al., 2020).
Most of the review articles on C. roseus have been published in 2007
on biosynthesis of MIAs and pharmacological properties (Hisiger and
Jolicoeur, 2007; Memelink and Gantet, 2007; MustafaVerpoorte, 2007;
Oudin et al., 2007; Pietrosiuk et al., 2007: Piovan and Filippini, 2007;
Zárate, and Verpoorte, 2007; Zhao and Verpoorte, 2007; Gajalakshmi
et al., 2013; Zhu et al., 2015; Nisar et al., 2016; Pan et al., 2016: Das and
Sharangi, 2017; Mahroug et al., 2007; Mishra and Verma, 2017; Sen­
bagalakshmi et al., 2017; Paarakh et al., 2019). The gaps of knowledge
2
Journal of Ethnopharmacology 284 (2022) 114647
on C. roseus are identified while preparing the comprehensive data and
presented for the speed-up drug discovery process. Furthermore, this
review also focuses on attracting the attention of scientific communities
about the widespread biological activities of this species of plant for its
better utilization prospects in the near future. Therefore, the present
review aims to provide comprehensive, critically analyzed and authentic
data on botanical description, traditional medicinal applications,
phytochemistry, biosynthesis, separation and analytical techniques,
pharmacological properties of extracts/active constituents and toxicity
of extracts/bioactive compounds of C. roseus till date.
An extensive survey of literature related to C. roseus was conducted
up to August 2021. The relevant literary works published on this plant
species related to botany, ethnomedicinal or traditional uses, phyto­
chemical constituents, pharmacological activities, and clinical studies
were collected from traditional Chinese medicine (TCM), Ayurveda, and
research articles across the world using a combination of the following
keywords: “C. roseus”, “bright eyes”, “cape periwinkle”, “graveyard
plant”, “Madagascar periwinkle”,“old maid”, “pink periwinkle”, “rose
periwinkle”, “medicinal uses of C. roseus”, “phytochemicals of
C. roseus”, “pharmacological activities of C. roseus”, “morphology,
description, taxonomy or botany of C. roseus”, synonyms as “Ammocallis
rosea”, “Lochnera rosea”, ‘Pervinca rosea”, ‘Vinca rosea”, “V. speciosa”,
various other accepted infra-specifics and synonyms of the plant were
also used but the maximum results were obtained from the above key­
words. The electronic databases used for the collection of relevant in­
formation of C. roseus include Google Scholar, Web of Science, Scopus,
PubMed, Science Direct, Taylor and Francis online, Springer Link, Na­
tional Center for Biotechnology Information (NCBI), Chemspider, and
SciFinder. The mentioned databases were used to collect detailed in­
formation about the common names, botanical characteristics,
geographical distribution, ethnobotany, phytochemistry, pharmacolog­
ical aspects, clinical studies, and toxicity on C. roseus. All presented
structures of C. roseus natural products/compounds were drawn using
ChemDraw Pro 8.0 software (PerkinElmer). The PubChem database was
used to check updated IUPAC names of phytochemicals reported for the
plant.
2. Botanical aspects
2.1. Taxonomy and classification
C. roseus plant species belongs to the order of ranking of Gentianales.
The member species of Apocynaceae family recorded to occur in trop­
ical, subtropical, and temperate biogeographic zones, mostly consisting
of deciduous trees, shrubs and herbs (Bihani, 2021). C. roseus is taxo­
nomically placed in the Kingdom Plantae, Class Magnoliopsida, Order
Gentianales, Family Apocynaceae and Genus Catharanthus (Integrated
Taxonomic Information System). There are about 359 genera in the
family and 9 (accepted) species for the genus Catharanthus (Plants of the
Whole Online). The various vernacular names of C. roseus are given in
Table 1. The synonyms of C. roseus include: Ammocallis rosea (L.) Small,
Lochnera rosea (L.) Rchb. ex Spach, Pervinca rosea (L.) Gaterau, Vinca
rosea L. and Vinca speciosa Salisb.
2.2. Morphology
C. roseus recorded to have various botanical characteristics that
differentiate this species from other plants within the genera and family.
The plant grows as a perennial herb or undershrub, up to 60 cm tall,
woody base, and milky latex sap (Allorge et al., 2015). The leaves are
petiolated, shape oblong, and texture leathery, whereas the arrangement
of leaves are opposite as well as alternate (Bertaccini, 2007; Junaid
et al., 2007). The flowers usually arise as single (Mishra et al., 2001),
attractive in colours which can be pink, white, or a mixture of both,
sometimes the pale pink with a dark violet dot in the center with many
hues and having spreading five petals (Singh et al., 2008). Based on
S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Table 1 flowers, roots) of this species have been employed for the treatment of
Vernacular names of C. roseus across the globe. diseases in various countries (Table 2). In Ayurveda, stem, leaves, and
S. Country Common name Reference roots of this plant species are employed to cure athelmintic, emetic,
N. digestive, hypotensive, laxative, sedative, stomachic, toothache, dia­
Bangladesh Nayantara (ক্যাথারান্থস) Islam et al. (2009a) betes, asthma, gastrointestinal ailments, and problems in female sexu­
Brazil Boa-noite, Congorca Arora et al. (2010) ality disorders (Rahman et al., 2004; Aslam et al., 2010; Gajalakshmi
Cook Islands Tiare-tupapaku-kimo (Arora et al. (2010) et al., 2013; Sagar et al., 2020). Few studies such as Nammi et al. (2003)
Dominica Caca poule Senbagalakshmi et al. and Al-Quteimat (2020) reported that eighteen leaves and some roots of
(2017)
French Pervenchede de Madagascar Hiruki & da Rocha
the plant boiled in a kettle of water taken in the Cook Islands and in West
Guiana (1986) Indies to control diabetes in the form of infused whiskey. C. roseus is also
Guatemala Chatilla Arora et al. (2010) utilised in folklore traditional medicine system to control menorrhagia,
Guyana, Periwinkle Marcone et al. (1997); toothache, high blood pressure, memory loss, and diarrhoea in various
Jamaica, USA Arora et al. (2010)
regions of India (Srinivasan et al., 2001; Govindasamy and Srinivasan,
India Ainskati, Billaganneru, Retna and Ethalsa
Nayantara, Nityakalyani, (2013) 2012; Rasineni et al., 2010). In India, mainly in the state of Odisha and
Periwinkle, Sadabahar, Arora et al. (2010) Assam, the fresh juice extracted from C. roseus leaves used to treat sting
Sadaphul, Ushamanjairi bites of wasp, whereas the root parts employed in the treatment of
Japan Nichinich-so, Nichinichi-so Arora et al. (2010) cancer. Besides, the C. roseus leaves is used to cure menorrhagia, bone
Kenya Maua Senbagalakshmi et al.
(2017); Arora et al.
disorders, hypertension, skin infections, diarrhoea/dysentery, dengue
(2010) fever, malaria, toothache, eye irritation, and respiratory problems in
Madagascar Madagascar periwinkle, Bright Arora et al. (2010) Thailand, Brazil, USA, Australia and China (Mehta et al., 2013; Kabesh
eye, Old maid, Pink perwinkle et al., 2015; Vipasha et al., 2016; Renjini et al., 2017). The European
Mexico Ninfa Arora et al. (2010)
people use this plant species for minor illnesses such as headaches and
Pakistan Sada-bahar Mishra and Verma
(2017) manage diabetes naturally. The roots have been used for the manage­
Peru Chavelita Arora et al. (2010) ment of hypertension due to their sedative and tranquilizing properties;
Philippines Atay-biya, Chichirica, Senbagalakshmi et al. similar compounds present in C. roseus also reported from different
Kantotan, Periwinkle, (2017); Arora et al. species of Rauwolfia as the tonic for general debility (Kumar et al. 2016a,
Tsitsirika (2010)
2016b). In Madagascar, leaves of C. roseus are employed as an emetic
Rodrigues Saponaire Gurib-Fakim et al.
Islands (1996) drug. Similarly, the underground root plant parts have been used as
Sri Lanka Minimal, Patti-poo Senbagalakshmi et al. laxative, and for curing toothache (Nejat et al., 2015). Young leaves
(2017); Arora et al. employed for the treatment of cramps in stomach and the decoction
(2010)
prepared from roots are used to removes the intestinal parasites in
Thailand Phaeng phoi farang, Phang- Arora et al. (2010)
puai- fa-rang Philippines. The leaves infusion employed for indigestion and dyspepsia
Venda Liluvha Arora et al. (2010) in Mauritius. In many countries, C. roseus floral extracts have been used
Vietnam Dua can for the treatment of dysmenorrhea (Indo-China), an eye-wash for infants
West Indies Brown man’s fancy, Arora et al. (2010) (Cuba and Jamaica), asthma/ respiratory problem (Bahamas), hyper­
Consumption bush, Old maid,
tension (Bermuda), malaria, menstrual problems and diaphoresis (Sur­
Periwinkle, Pink flower, Ram
goat rose, Red rose, Sailor’s inam and Caribbean) (Aslam et al., 2010; Senbagalakshmi et al., 2017).
flower, White tulip The extracts or infusions of leaves of C. roseus is used by the people of
Uganda to cure ulcers associated with digestive tracts, whereas the dried
leaf powder with milk helpful for the treatment of full-grown abscesses
variation in flower colour, there are two varieties of C. roseus. The one in Botswana (Naeem et al., 2019). The root decoction is also used to cure
producing pink flower is called the “Rosea” and the other having the dysmenorrhea in Togo (Senbagalakshmi et al., 2017). In China, this
white flowers is known as “Alba” (Sagar et al., 2020; Al-Quteimat, 2020) species is widely used to cure blood cancer, cough and Brill Symmers
(Fig. 1). Fruits comprised of two narrow cylindrical follicles having (Senbagalakshmi et al., 2017). Correspondingly, Central and South
numerous seeds. America’s people accepted this herb as the plant species for lungs
congestion and throat infections (Don, 1999; Ross, 2003; Das and
2.3. Geographic distribution Sharangi, 2017; Mishra and Verma, 2017; Renjini et al., 2017; Senba­
galakshmi et al., 2017). The tea prepared from leaves of the plant has
The natural wild habit of C. roseus is Madagascar and in the due been used to treat the both mouth and gastric ulcers in Asian countries
passage of time, the plant got distributed across the globe either as an (Rambhai et al., 2019; Tabuti et al., 2003).
ornamental plant or as introduced species for medicinal purposes (Gupta
et al., 2007: Aruna et al., 2015; Falcão et al., 2017). The wide range of 4. Phytochemistry
distribution in various countries recorded for this species is presented in
Table 1. Tong et al. (2011) reported C. roseus as an evergreen herbaceous 4.1. Biosynthesis and synthetic biology advances
ornamental plant of tropical countries. Total nine species reported under
the genus, out of which 7 species known from Madagascar; one from Synthetic biology play an important role in biosynthesis of MIAs and
India and Sri Lanka, while one species is cultivated in China (El-Ghazaly, bisindole alkaloids via combination of various disciplines including
1990; Allorge et al., 2015). C. roseus is more commonly known as molecular biology, molecular engineering, mathematics and physics to
Madagascar periwinkle. It has a side effects on skin such as warm design and build novel proteins, genetic circuits and metabolic networks
prickling and hallucinations (Das and Sharangi, 2017). (McDaniel and Weiss, 2005). Biosynthesis of assembly of these MIAs and
bisindole alkaloids is a multi-step and enzyme-catalyzed process where
3. Traditional uses and ethnopharmacology more than one substrate is converted into more complex metabolites in
parts of C. roseus (Fig. 2). The leaf epidermis is enriched with synthetic
C. roseus has been of prime importance in the traditional medicine enzymes where most of the MIAs and bisindole alkaloids biosynthesis
systems and has wide therapeutic applications for many centuries due to are expressed. 3α (S)-strictosidine (105) is an important intermediate in
its unpleasant side effects. The different plant parts (leaves, stems, biosynthesis of most of MIAs, which is a condensation product of

3
S. Kumar et al.
Fig. 1. Morphology of C. roseus, (A) habit of the whole plant, (B) close view of stem and leaves, (C) root parts, (D–E) close view of flowers, (F) fruits, (G) seeds.
secologanin and tryptamine. Vindoline (27) and catharanthine (55),
which are produced by compound 105 via multi-step enzymatic reac­
tion, can form α-3, 4-anhydrovinblastine by the condensation reaction.
In biosynthesis of bisindole alkaloids like compounds 111 and 112
formed by togather combination of compounds 55 and 27 are modified
and converted into iminium ions. Both iminium ions and 3′ ,4′ -anhy­
drovinblastine (117) are interconvertible to each other and final
biosynthetic product is compound 111. Furthermore, compounds 112
and leurosine (125) are synthesized from compound 111 but convertible
enzymes involved during the biosynthesis are unknown. Only 26 genes
involved in the assembly of these two bisindole alkaloids are known, two
key reactions have eluded characterization to complete the documen­
tation of the compound 111 pathway in C. roseus (Memelink and Gantet,
2007; Zhu et al., 2015; Mahroug et al., 2007; Quan et al., 2019).
Many transcription factors are involved in the regulation of MIAs
biosynthesis, such as apetala2/ethylene responsive factor (AP2/ERF)
and WRKY. The illustration of biosynthetic pathways have laid a basis
for the study of synthetic biology. Recently, compounds 27 and 105
have been synthesized in heterologous hosts saccharomyces cerevisiae.
Research about synthetic biology and the regulation mechanisms will
provide guidance for the production and development of MIAs and
bisindole alkaloids drugs in C. roseus (Oudin et al., 2007; Zárate, and
Verpoorte, 2007; Pan et al., 2016).
Total of 345 compounds including alkaloids, phenolics and volatile
compounds have been categorized and summarized in the review. The
bisindole alkaloids (compounds 111), 112, 121), vincovalinine (137),
vincovalicine (138), vincadioline (139)) and MIAs (compounds 27, 55,
vinpocetine (52), trichophylline (54), vincarodine (56), and pleiocarp­
amine (57)), are characteristic as a major/characteristic compounds
which were isolated, identified, and chemically characterized from
different parts especially leaves of C. roseus (Rao and Ahmed, 2014;
Lawal et al., 2015; Nisar, 2017a, 2017b; Kumar et al., 2018a). Most of
flavonoids, phenolic acids, triterpenes and sterols have been identified
and characterized by liquid chromatography–mass spectrometry
(HPLC-MS) and liquid chromatography with tandem mass spectrometry
(HPLC-MS/MS), whereas gas chromatography–mass spectrometry
(GC-MS) are used for the analysis of volatile chemical constituents from
extracts of C. roseus parts (Jeong and Lim, 2018; Barrales-Cureño et al.,
4
Journal of Ethnopharmacology 284 (2022) 114647
2019). The compounds and their corresponding classes are presented in
Table 3.
4.2. Monoterpene indole alkaloids (MIAs)
Total of 106 MIAs (1–106) and three indole derivatives (107–109)
and one piperidine derivatives (110) have been reported from different
parts (leaves, roots, stems) of C. roseus and tissue culture of leaves, roots,
and stems (Wang et al., 2012a). Biosynthetic pathways showed that the
combination of tryptophan and serotonin produced compound 105 and
further many enzymatic reactions synthesized an intermediate or pre­
cursor either vincoside (96) or compound 105 or both simultaneously.
Compounds 96 and 105 synthesized a corynantheine class of interme­
diate (97–104 and 106) due to cyclization of tepene ring in different
manner which were detected and isolated from leaves (O’Connor and
Maresh, 2006; O’Connor et al., 2012). Corynantheine is an intermediate
for synthesis of sarpagine (61–71), ajmaline (72–75), reserpine (76–79)
and ajmalicine (80–95) classes of MIAs. The cyclization and rear­
rangement of corynantheine generated three classes of MIAs namely
vincadifformine (1–26), tabersonine (27–51) and catharanthine (52 and
53). Oxidation and reduction at C-2 and C-3 by both directions i.e. front
and back with the help of enzymes produced many MIAs of classes
vincadifformine (1) and tabersonine (6). Similar circumstance occurred
with the C-6 and C-7 tabersonine (6–26) and vindoline (27–51) with
enzymatic reduction (SN 15) oxidation (16–20) and epoxidation
(16–20) of double bond of terpene. The cyclization of terpenine moiety
is synthesizes a variety of complex skeletons 46–52 and 54–60). Most of
the sarpagine (65), ajmaline (72), ajmalicine (80) and reserpine (78) of
MIAs have been reported from C. roseus and Rauwolfia species (Kumar
et al., 2016a, 2016b, Baig et al., 2021) (Fig. 3Aand 3B).
4.3. Bisindole alkaloids
A total of 35 (111–145) bisindole alkaloids have been reported in
different parts of C. roseus and its suspension cell cultures (Wang et al.,
2012b, 2014) (Fig. 4A and4B). Biosynthetically, the bisindole alkaloids
(like (111) and 112) synthesized via combination of two different and
same moieties with C–C bond formation at C-10 with C-16′ like in
S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Table 2
Ethnomedicinal applications of different herbal preparations of plant parts of C. roseus in world-wide.
S. Plant Preparation Ethnomedicinal use Country Reference
N. part

1. Whole Decoction Muscle relieve pain, depression of CNS, wasps stings India Ross (2003); Don (1999) Das and Sharangi
plant (2017); Renjini et al. (2017); Senbagalakshmi
et al. (2017)
Decoction Nose bleed, bleeding gums, mouth ulcers, sore throats India Senbagalakshmi et al. (2017)
The hot water extract taken Cancer India Senbagalakshmi et al. (2017); Aslam et al.
orally (2010)
The plant was boiled to Stop bleeding Hawaii (Das and Sharangi, 2017; Renjini et al., 2017;
make a poultice Aslam et al., 2010)
Decoction Diabetes, cancer Senbagalakshmi et al. (2017)
Hot water extract of root Diabetes mellitus Brazil Senbagalakshmi et al. (2017); Aslam et al.
bark, dried entire plant (2010)
Hot water extract of dried Diabetes Thailand Senbagalakshmi et al. (2017); Aslam et al.
entire plant (2010)
Decoction Kidney problems, diabetes, arthritis Marshall Senbagalakshmi et al. (2017); Aslam et al.
Islands (2010)
Extract Cure stomach ulcer, sedative, and anticancer Japan Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Hot water extract of dried Antigalactagogue England Aslam et al. (2010)
entire plant
Juice For high blood pressure Dutch Senbagalakshmi et al. (2017)
Hot water extract of entire Antigalactogogue Vietnam Senbagalakshmi et al. (2017); Aslam et al.
plant (2010)
Hot water extract of entire Antigalactagogue France (Senbagalakshmi et al. (2017); Aslam et al.
plant (2010)
Decoction Used for diabetes Cuba (Das and Sharangi, 2017; Renjini et al., 2017;
Aslam et al., 2010)
Hot water extract of dried Cancer, heart disease, and leishmaniasis Peru Senbagalakshmi et al. (2017); Aslam et al.
entire plant (2010)
Decoction High blood pressure Venda Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Gargling with an infusion Relieve pain from a sore throat, laryngitis, and chest USA Das & Sharangi (2017); Aslam et al. (2010)
of the plant complaints and ease luncogestion inflammation
Extract Eye irritation and infection Caribbean Senbagalakshmi et al. (2017)
Decoction Diabetes and sedative Caribbean Senbagalakshmi et al. (2017)
Decoction Diabetes Myanmar Aslam et al. (2010)
decoction Diabetes Malaysia Das and Sharangi (2017)
Decoction of dried entire Diabetes mellitus, liver diseases Taiwan Das and Sharangi (2017)
plant
2. Aerial Infusion Orally for diabetes Kenya Senbagalakshmi et al. (2017); Aslam et al.
parts (2010)
Hot water extract Menstrual regulator Vietnam Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Hot water extract Menstrual regulator, malaria China Renjini et al. (2017); Aslam et al. (2010)
3. Stem Hot water extract Diabetes West Indies Das and Sharangi (2017); Renjini et al.
(2017); Aslam et al. (2010)
Decoction Dysmenorrheal Indonesia Senbagalakshmi et al. (2017); Aslam et al.
(2010)
4. Leaves Infusion Menorrhagia, Toothache, memory loss, blood circulation India Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Hot water extract of leaves Diabetes mellitus, amenorrhea, Philippines Das and Sharangi (2017); Renjini et al.
(2017); Aslam et al. (2010)
Decoction Regulating blood sugar, reducing toothaches, cardiovascular Sri Lanka Senbagalakshmi et al. (2017)
diseases, soothing insect bites, improving memory and
circulation, healing wounds
Hot water extract Diabetes England Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Decoction Vomitive France Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Hot water extract Combat primary inertia in childbirth; diabete Dominica (Senbagalakshmi et al., 2017; Aslam et al.,
2010)
Hot water extract Menorrhagia and diabetes. Australia (Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Expressed juice together Anemia. Myanmar Senbagalakshmi et al. (2017); Aslam et al.
with the same amount of (2010)
honey.
Crush fresh leaves Ulcers, inflammation Myanmar Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Decoction Traditionally used for diabetes Spain Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Decoction Astringent, diuretic, cough remedy China Renjini et al. (2017), Aslam et al. (2010)
Hot water extract Diabetes, rheumatism, Mozambique Senbagalakshmi et al. (2017); Aslam et al.
(2010)
juice Indigestion, dyspepsia Mauritius Das and Sharangi (2017); Aslam et al., 2010)
(continued on next page)

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S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Table 2 (continued )
S. Plant Preparation Ethnomedicinal use Country Reference
N. part

Decoction Menorrhagia, rheumatism Africa Das and Sharangi (2017)

Decoction Toothache, fever, skin diseases, diuretic. India Chandran & Suarsana et al. (2015)
5. Dried Hot water extract is taken Hodgkin’s disease India Aslam et al. (2010)
leaves orally
Decoction Diabetes, hypertension, cancer Cook Islands Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Hot water extract Diabetes Kenya Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Hot water extract Diabetes Guatemala Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Hot water extract Diabetes Jamaica Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Hot water extract Menorrhagia, diabetes Africa Renjini et al. (2017)
Decoction Diabetes mellitus Europe Senbagalakshmi et al. (2017); Aslam et al.
(2010)
6. Flower An extract of the flower Eyewash for the eyes of infants Rodriguez Senbagalakshmi et al. (2017); Aslam et al.
Islands (2010)
Extract Eye wash for the eyes of infants Jamaica Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Decoction Asthma, tuberculosis, flatulence Bahamas Das and Sharangi (2017); Renjini et al. (2017)
7. Dried Hot water extract Diabetes Pakistan Aslam et al. (2010)
ovules
8. Root The extract is taken orally Menorrhagia India Senbagalakshmi et al. (2017)
Decoction Tonic, stomachic India Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Hot water extract of dried Emmenagogue Philippines Das and Sharangi (2017)
root used orally
Hot water extract of root Abortion, emmenagogue, dysmenorrheal with scanty flow Philippines Das and Sharangi (2017); Renjini et al.
abortion (2017); Aslam et al. (2010)
Infusion Diabetes Mexico Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Decoction Purgative, vermifuge, depurative, hemostatic; toothache France Aslam et al. (2010)
remedy
Hot water extract Hypertensive, febrifuge Mozambique Aslam et al. (2010)
Decoction Vomitive, purgative, vermifugl, depurative, hemostatic and Madagascar Das and Sharangi (2017)
toothache remedies
9. Dried Decoction Fevers, malaria. Brazil Aslam et al. (2010)
root
Decoction Stomach problem Kenya Senbagalakshmi et al. (2017); Aslam et al.
(2010)
Water extract Venereal disease Venda Aslam et al. (2010)
10. Root Hot water extract Febrifug Australia Aslam et al. (2010)
bark

epidermis of C. roseus leaf. They are formed by the combination of
compounds 27 and 55 due to nucleophilic attack on indole moiety
(Palaniappan et al., 2020). Alkaloids 111–115 were produced due to
substitution or addition of CH3, OH, H- on terpene moiety of cathar­
anthine (55) at C-20′ as well as N- on vindole moiety. Leurosine-Nb-­
oxide (126), pseudovinblastinediol (116), deacetoxyvinblastine
(desacetoxyvinblastine) (118), leurosidine-N-oxide (124) and leur­
ocolornbine (132) have been reported to have additional OH of the
catharanthine (55) moiety. 3′ ,4′ -Anhydrovinblastine (117) yielded due
to hydrogenation of terpene of the catharanthine moiety. 17-Deacetox­
yvinblastine N’b-oxide ( 119), 20′ -deoxyvinblastine N’b-oxide (120)
and leurosidine-N-oxide (124) formed due to oxidation of terpene on
N-atom of the catharanthine moiety. Compound 121 is regarded as a
type of bisindole bisindole alkaloid formed due to the substitution of
CH3CO with NH2 in vindoline (27) moiety. Leurosine (125), deacetox­
yleurosine (desacetoxyleurosine) (128), 5′ -oxoleurosine (129),
19′ -oxoleurosine (130) and vinleurosine (131) have been formed due to
double bond terpene epoxidation of catharanthine moiety. Similarly, all
bisindole alkaloids have been synthesized in different parts of C. roseus
especially leaves and suspension cell cultures via oxidation, epoxidation
and dehydrogenation (Frasci et al., 2000; Jacobs et al., 2004; Yang et al.,
2011, Zhao et al., 2013; Zhang et al., 2013; Baig et al., 2021).
4.4. Flavonoids and phenolic acids
A total of 34 flavonoids (146–179) and nine phenolic acids
(180–188) have been identified from different plant parts of C. roseus
(Fig. 5A-C). The quercetin-3-O-arabinoside (147), rutin (148), quer­
cetin-3-O-(2,6-di-O-rhamnosyl) galactoside (149), quercetin-3-O-(2,6-
di-O-rhamnosyl)glucoside (150) and quercetin-3-O-(2,6-di-O-rhamno­
syl-glucoside) (151) were regarded as hexoside derivatives of quercetin
in hydro-alcoholic extracts of the leaves of this plant. Similarly,
kaempferol ( 151) and its glucosides, kaempferol-3-O-(6-O-rhamnosyl
glucoside (153), kaempferol-3-O-(2,6-di-O-rhamnosyl) galactoside
(154) and kaempferol-3-O-(2,6-di-O-rhamnosyl) glucoside (155) have
identified in this plant by HPLC-MS/MS. Isorhamnetin-3-O-(2,6-di-O-
rhamnosyl) galactoside (156), isorhamnetin-3-O-(2,6-di-O-rhamnosyl-
galactoside (isomer) (157), isorhamnetin-3-O-(2,6-di-O-rhamnosyl)
glucoside (158), isorhamnetin-3-O-(6-O-rhamnosyl) glucoside (159),
isorhamnetin-3-O-(6-O-rhamnosyl) galactoside (160) and isorhamnetin-
3-O-(6-O-rhamnosyl) hexoside (161) were characterized as hexosides of
isorhamnetin, whereas, syringetin-3-O-robinobioside ( 162) was pre­
sented as glucoside of syringetin. 3′ ,4′ -di-O-methylquercetin-7-O-
[(4′′ →3′′′ )-2′′′ ,6′′′ ,10′′′ ,14′′′ -tetramethylhexadec-13′′′ -ol-14′′′ -enyl]-β-D-
glucopyranoside (163), 4′ -O-methylkaempferol-3-O-[(4′′ →3′′′ )-
2 ,6 ,10 ,14 -tetramethylhexadecan-13′′′ -olyl]-β-D-glucopyranoside
′′′ ′′′ ′′′ ′′′

(164), 3′ ,4′ -di-O-methylbutin-7-O-[(6′′ →1′′′ )-3′′′ ,11′′′ -dimethyl-7′′′ -

methylenedodeca-3′′′ ,10′′′ -dienyl]-β-D-glucopyranoside (165) and 4′ -O-

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S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Fig. 2. Biosynthetic pathways of different classes of Catharanthus indole alkaloids. TDC: tryptophan decarboxylase, LAMT: Loganic acid-O-methyltransferases, SLS:
secologanin synthase, STR: strictosidine synthase, GO: geissoschizine oxidase (Redox115), (Redox216), SAT: stemmadenine O-acetyltransferase, ASO: O-acetyl­
stemmadenine oxidase, GS: geissoschizine synthase, HL3: hydrolase 3, HL4: hydrolase 4. HL1: hydrolase 1, T16H: tabersonine 16-hydroxylase, T3O: tabersonine 3-
oxidase, 16OMT: 16-hydroxytabersonine 16-O-methyltransferase, T3R: tabersonine 3-reductase, NMT: 2,3-dihydro-3-hydroxytabersonine-N-methyltransferase, D4H:
desacetoxyvindoline 4-hydroxylase, DAT: deacetylvindoline 4-O-acetyltransferase.

methylbutin-7-O-[(6′′ →1′′′ )-3′′′ ,11′′′ -dimethyl-7′′′ -hydroxymethylenedo-
decanyl]-β-D-glucopyranoside (166) were a special type of flavonoids
formed due to a combination of hexoside and terpene in the methanol
extract of hairy roots. Petunidin-3-O-glucoside (174), petunidin-3-O-(6-
O-p-coumaroyl) glucoside (177), malvidin-3-O-glucoside (175),
malvidin-3-O-(6-O-p-coumaroyl) glucoside (179) hirsutidin-3-O-gluco­
side (176) and hirsutidin-3-O-(6-O-p-coumaroyl) glucoside (178) have
been reported as hexoside of petunidin (171), malvidin (170) and hir­
sutidin (172), respectively. Pelargonidin (167), cyaniding (167), peo­
nidin (169), 170–172 and delphinidin ( 173) were considered as

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S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Table 3
Various classes of phytochemical from C. roseus.
S. Class Sub-class Compound Reference
N.

1 Alkaloids MIAs Vincadifformine Ramirez and García-Rubio (2003); Jacobs et al. (2004); Levac
2 Minovincinine et al. (2008); Arora et al. (2010); Pan et al. (2010); Soares et al.
3 Echitoveniline (2012); Wang et al. (2014); Wesołowska et al. (2016); Alam et al.
4 Echitovenidine (2017); Kumar et al. (2018a); Kumar et al. (2018b); Zhu et al.
5 Echitovenine (2018); da Silva e Silva et al. (2019); Koel et al. (2020)
6 Tabersonine
7 11-Hydroxytabersonine
8 11-Methoxytabersonine
9 Nor 4-hydroxytabersonine
10 16-methoxytabersonine
11 16-Hydroxytabersonine
12 19-Hydroxy-11-methoxytabersonine
13 19-Acetoxy-11-hydroxytabersonine
14 19-Acetoxy-11-methoxytabersonine
15 Minovincine
16 Lochnericine
17 19(S)-epimisiline
18 Lochnerinine
19 Horhammericine
20 Horhammerinine
21 Lochneridine
22 20-epi-Lochneridine
23 Akuammicine
24 Vinervine
25 Cathaphylline
26 Tubotaiwine (Dihydrocondylocarpine)
27 Vindoline
28 Vindorosine
29 Deacetylvindoline
30 Deacetylvindoline
31 4-O-deacetyl-N,-demethylvindolin
32 4-O-deacetylvindorosine
33 N,-demethyldeacetylvindorosine
34 16-O-Demethylvindoline
35 16-O,N,-bisdemethyl-4-O -deacetylvindolin
36 16-Methoxy-2,3-dihydro-3-hydroxytabersonine
37 Bannucine
38 Vindolidine
39 Vincoline
40 Kitraline
41 Dihydrovindoline
42 Venalstonine
43 Epivindolinine (vindolinine)
44 5(S)-Hydroxy-14,15-dihydrovindolinine
45 Vindolinine-Nb-oxide (9-Epivindolinine-Nb-oxide)
46 Venalstonidine
47 11-Methoxy-2,16-di-hydrotabersonine
48 2,3-Dihydrotabersonine
49 Cathovaline
50 Deacetylcathovaline
51 Hydroxycathovaline
52 Vinpocetine
53 Vincamine
54 Trichophylline
55 Catharanthine
56 Vincarodine
57 Pleiocarpamine
58 Coronaridine
59 Stemmadenine
60 Dehydrosecodine
61 Akuammiline
62 Desacetylakuammiline
63 10-Hydroxy-deacetylakuammiline
64 10-O-methylsarpagine (Lochnerine)
65 Sarpagine
66 Perivine
67 Pericyclivine
68 Vellosiminol
69 21-hydroxycyclolochnerine
70 10-Hydroxydeformodihydropseudoakuammigine 10-O-L-
arabinopyranoside
71 10-Hydroxycathafoline 10-O-larabinopyranoside
72 Ajmaline
(continued on next page)

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S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Table 3 (continued )
S. Class Sub-class Compound Reference
N.

73 10-Hydroxynortetraphyllicine
74 Norseredamine
75 Tetraphyllicine
76 Pseudoyohimbine
77 Yohimbine
78 Reserpine
79 3,4,5,6-tetradehydroyohimbine
80 Ajmalicine
81 3-Isoajmalicine
82 Akuammigine
83 Tetrahydroalstonine
84 19-Epiajmalicine
85 3-Iso-19-epi-ajmalicine
86 3-Epi-19-epiajmalicine
87 3-Epiajmalicine
88 Reserpiline
89 7-Hydroxyindolenineajmalicine
90 Alstonine
91 Cathenamine
92 Pseudoindoxyl ajmalicine
93 Strictosidine lactam
94 Mitraphylline
95 Serpentine
96 Vincoside
97 Dihydrositsirikine
98 Sitsirikine
99 Isositsirikine (16R,19E)
100 Isositsirikine (16R,19Z)
101 O-Acetylstemmadenin (strychnos)
102 Vallesiachotamine
103 17-Isovallesiachotamine
104 Geissoschizine
105 Strictosidine (3α (S)-strictosidine)
106 Anthirine
107 β-Carboline
108 n,n-dimethyltryptamine
109 Tryptamine,Nb-acetyl
110 Iochrovicine
111 Bisindole Vinblastine Frasci et al. (2000); Jacobs et al. (2004); Yang et al. (2011), Wang
alkaloid et al. (2012a); Wang et al. (2012b); Zhao et al. (2013); Zhang et al.
112 Vincristine (2013); Alam et al. (2017); Jeong and Lim (2018); Kumar et al.
113 4′ -Deoxyvinblastine (2018a); Kumar et al. (2018b)
114 Demethylvinblastine
115 Deoxyvincaleukoblastine B
116 Pseudovinblastinediol
117 3′ ,4′ -Anhydrovinblastine
118 Deacetoxyvinblastine (Desacetoxyvinblastine)
119 17-Deacetoxyvinblastine N’b-oxide
120 20′ -Deoxyvinblastine N’b-oxide
121 Vindesine
122 Catharine
123 Leurosidine
124 Leurosidine-N-oxide
125 Leurosine
126 Leurosine-Nb-oxide (Pleurosine)
127 Isoleurosine
128 Deacetoxyleurosine (Desacetoxyleurosine)
129 5′ -Oxoleurosine
130 19′ -Oxoleurosine
131 Vinleurosine
132 Leurocolornbine
133 Roseadine
134 Vincathicine
135 Catharinine (Vinamidine)
136 Vincovaline
137 Vincovalinine
138 Vincovalicine
139 Vincadioline
140 Vindolicine
141 Catharanthamin
142 Vingramine
143 Vinorelbine
144 Vinrosidine
145 Methylvingramine
146 Flavonoid Flavonol Quercetin
(continued on next page)

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S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Table 3 (continued )
S. Class Sub-class Compound Reference
N.

147 Quercetin-3-O-arabinoside Piovan and FilippiniFavretto (1998); Kessler et al. (2003); Choi
148 Rutin et al. (2002); Piovan and Filippini (2007); Yang et al. (2008);
149 Quercetin-3-O-(2,6-di-O-rhamnosyl) galactoside Chung et al. (2009); He et al. (2010); Ferreres et al. (2011); Rao
150 Quercetin-3-O-(2,6-di-O-rhamnosyl) glucoside and Ahmed (2014); Nisar (2017a); Nisar (2017b); Lahare et al.
151 Quercetin-3-O-(2,6-di-O-rhamnosyl-glucoside) (2020); Yu et al. (2021)
152 Kaempferol
153 Kaempferol-3-O-(6-O-rhamnosyl) glucoside
154 Kaempferol-3-O-(2,6-di-O-rhamnosyl) galactoside
155 Kaempferol-3-O-(2,6-di-O-rhamnosyl) glucoside
156 Isorhamnetin-3-O-(2,6-di-O-rhamnosyl) galactoside
157 Isorhamnetin-3-O-(2,6-di-O-rhamnosyl) galactoside (isomer)
158 Isorhamnetin-3-O-(2,6-di-O-rhamnosyl) glucoside
159 Isorhamnetin-3-O-(6-O-rhamnosyl)glucoside
160 Isorhamnetin-3-O-(6-O-rhamnosyl) galactoside
161 Isorhamnetin-3-O-(6-O-rhamnosyl) hexoside
162 Syringetin-3-O-robinobioside
163 3′ ,4′ -di-O-methylquercetin-7-O-[(4′ ′ →3′′′ )-2′′′ ,6′′′ ,10′′′ ,14′′′ -
tetramethylhexadec-13′′′ -ol-14′′′ -enyl]-β-D-glucopyranoside
164 4′ -O-methylkaempferol-3-O-[(4′ ′ →3′′′ )-2′′′ ,6′′′ ,10′′′ ,14′′′ -
tetramethylhexadecan-13′′′ -olyl]-β-D-glucopyranoside
165 3′ ,4′ -di-O-methylbutin-7-O-[(6′ ′ →1′′′ )-3′′′ ,11′′′ -dimethyl-7′′′ -
methylenedodeca-3′′′ ,10′′′ -dienyl]-β-D-glucopyranoside
166 4′ -O-methylbutin-7-O-[(6′ ′ →1′′′ )-3′′′ ,11′′′ -dimethyl-7′′′ -
hydroxymethylenedo-decanyl]-β-D-glucopyranoside
167 Anthocyanin Pelargonidin
168 Cyanidin
169 Peonidin
170 Malvidin
171 Petunidin
172 Hirsutidin
173 Delphinidin
174 Petunidin-3-O-glucoside
175 Malvidin-3-O-glucoside
176 Hirsutidin-3-O-glucoside
177 Petunidin-3-O-(6-O-p-coumaroyl) glucoside
178 Hirsutidin-3-O-(6-O-p-coumaroyl) glucoside
179 Malvidin-3-O-(6-O-p-coumaroyl) glucoside
180 Phenolic Phenolic acid Gallic acid Kroes et al. (1992); Kratz et al. (2008); Li et al. (2020); Nisar
acid (2017a)
181 Ferulic acid
182 Caffeic acid
183 Benzoic acid
184 Vanillic acid
185 Coniferyl aldehyde
186 4-O-Caffeoylquinic acid
187 5-O-Caffeoylquinic acid
188 3-O-Caffeoylquinic acid
189 Volatile Alkane n-Dodecane (Brun et al. (2001); Pandey-Rai et al. (2006); De Pinho et al.
compound (2009); Lawal et al. (2015); Fitrianto et al. (2020); Syeda and
190 n-Tridecane Riazunnisa (2020)
191 n-Tetradecane
192 n-Pentadecane
193 Heptadecane
194 Octadecane
195 Nonadecane
196 Eicosane
197 Heneicosane
198 2,6,11-Trimethyl dodecane
199 Docosane
200 Tricosane
201 Tetracosane
202 Dotriacontane
203 Tetracontane
204 Alkene n-Hexadecene
205 Heneicosene
206 Docosene
207 (Z)-9-Tricosene
208 (Z)-β-Ocimene
209 Myrcene
210 (E,E)-α-Farnesene
211 3-Phenyldodecane
212 Limonene
213 β-Phellandrene
214 γ-Terpinene
215 β-Bisabolene
(continued on next page)

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S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Table 3 (continued )
S. Class Sub-class Compound Reference
N.

216 Germacrene D
217 γ-Cadinene
218 Trans-α-Bergamotene
219 α-Pinene
220 β-Pinene
221 Aldehyde Heptanal
222 Octanal
223 Nonanal
224 Decanal
225 Undecanal
226 Dodecanal
227 Tetradecanal
228 Pentadecanal
229 Hexadecanal
230 (E)-2-Octenal
231 (E)-2-Nonenal
232 (E)-2-Decenal
233 (E)-2-Undecenal
234 (E,E)-2,4-Hexadienal
235 (E,E)-2,4-Heptadienal
236 (E,E)-2,4-Decadienal
237 (E,E)-2,4-Dodecadienal
238 (E,Z)-2,4-Decadienal
239 (E, Z)-2,6-Nonadienal
240 Neral
241 6,6-Trimethylcyclohexen-1-acetaldehyde
242 Perilla aldehyde
243 β-Cyclocitra
244 Safranal
245 Benzaldehyde
246 4-Methylbenzaldehyde
247 Ethylbenzaldehyde
248 Ketone 2-Methyl 4-heptanone
249 Pentadecane-2-one
250 Hexahydrofarnesylacetone
251 Heptenone
252 3-Octen-2-one
253 Isophorone
254 β-Pulegone
255 (E)-Ionone-5,6-epoxide
256 (E)-α-Ionone
257 (E,E)-Octa-3,5-dien-2-one
258 (E,Z)-Octa-3,5-dien-2-one
259 6-Methyl-3,5-heptadien-2-one
260 (E)-β-Ionone
261 (E,E)-Pseudoionone
262 Megastigmatrienone
263 (E)-Damascenone
264 2,6-bis(1,1-dimethylethyl)- 2,5-Cyclohexadiene-1,4-dione
265 6-Methyl hept-5-ene-2-one
266 2,2,6-Trimethylcyclohexanone
267 6-Methyl-5-hepten-2-one
268 Geranylacetone
269 Farnesylacetone
270 Davanone
271 Alcohol Dodecyl alcohol
272 Hexadecanol
273 Eicosan-1-ol
274 Phytol
275 Citronellol
276 9-Octadecen-1-ol
277 Nerol
278 Geraniol
279 (E,E)-Farnesol
280 (Z,Z)-Farnesol
281 (E,Z)-Farnesol
282 Iso-Dihydro carveol
283 Isopulegol
284 Linalool
285 Isophytol
286 1-[(E)-1-Hexenyl]-cyclohexanol
287 Trans-Sabinene hydrate
288 α-Eudesmol
289 γ-Eudesmol
290 Terpinen-4-ol
(continued on next page)

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S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Table 3 (continued )
S. Class Sub-class Compound Reference
N.

291 α-Terpineol
292 Cedrol
293 Widdrol
294 Elemol
295 2,6-di-tert -butyl-p-cresol
296 2-Hydroxy 5-methoxy p-cymene
297 (E)-Anethole
298 Eugenol
299 Methyl eugenol
300 Methyl salicylate
301 Lanosterol
302 β-Sitosterol
303 α-Amyrin
304 Acid Caproic acid
305 Caprylic acid
306 Nonanoic acid
307 Capric acid
308 Undecanoic acid
309 Lauric acid
310 Tridecanoic acid
311 Myristic acid
312 Pentadecanoic acid
313 Palmitic acid
314 Stearic acid
315 Hexadecenoic acid
316 Linoleic acid
317 Octadecenoic acid
318 Dehydroabietic acid
319 Methyl heptanoate
320 Methyl caprylate
321 Methyl undecanoate
322 Methyl myristate
323 Methyl pentadecanoate
324 Methyl palmitate
325 Methyl 9-tridecenoate
326 Hexatrienoic acid methyl ester
327 Linoleic acid methyl ester
328 Linolenic acid methyl ester
329 Oleic acid ethyl ester
330 Linolenic acid ethyl ester
331 Isoamyl laurate
332 Ester Dodecyl acetate
333 Hexadecyl acetate
334 Neryl acetate
335 (E,E)-Farnesyl acetate
336 α-Terpinyl acetate
337 α-Tocopherol
338 Acetate Dihydroactinidiolide
339 (Z)-Linalool oxide
340 (E)-Linalool oxide
341 Theaspirane B
342 Theaspirane A
343 Edulane II
344 1,8-Cineole

anthocyanins and formed due to the substitution of hydrogen from O–H
with an alkyl group. Gallic acid (180), ferulic acid ( 181), caffeic acid
(182), benzoic acid (183), vanillic acid (184), coniferyl aldehyde (185),
4-O-caffeoylquinic acid (186), 5-O-caffeoylquinic acid ( 187) and 3-O-
caffeoylquinic acid (188) have been identified and characterized as
phenolic acids especially gallic and caffeic acid derivatives from the
different extracts of leaves (Kessler et al., 2003; Piovan and Filippini,
2007; Yang et al., 2008; Ferreres et al., 2011; Rao and Ahmed, 2014;
Nisar, 2017a, 2017b).
4.5. Volatile compounds
Total of 156 volatile compounds including alkanes (189–203), al­
kenes (204–220), aldehydes (221–247), ketones (248–270), alcohols
(271–304), acids (305–319), esters (320–332), acetates (333–338),
lactones (339–344) were summarized from different plant parts
especially leaves of C. roseus (Brun et al., 2001; Pandey-Rai et al., 2006;
De Pinho et al., 2009; Lawal et al., 2015; Fitrianto et al., 2020; Syeda and
Riazunnisa, 2020). All these volatile compounds reported from C. roseus
are presented in Fig. 6A and B.
5. Quality control/quality assurance: separation and analysis
techniques
The separation and analysis techniques such as thin-layer chroma­
tography (TLC), high-performance thin-layer chromatography (HPTLC),
high-performance liquid chromatography (HPLC), HPLC-MS, HPLC-MS/
MS, gas chromatography (GC) and GC-MS have been evaluated herbal
products for quality control (Kumar et al., 2016a, 2016b). Several
analytical methods have been reported for separation, qualitatitation
and characterization of bioactive compounds from complex matrix such
plant extracts mainly ethanolic and methanolic as well as alkaloidal

12
S. Kumar et al.
Fig. 3A. Structures of monoterpene indole alkaloids (1–59) from C. roseus.
fractions of plant parts of C. roseus to speed up drug discovery process
and quality control/quality assurance. The analytical methods and
separation techniques and their methodology are presented in Table 4.
The analytical approaches classified as (i) qualitative analysis which
aims metabolic profiling, to display a large number of primary and
secondary metabolites from the extracts, (ii) quantitative analysis of
targeted phytochemicals from complex matrix (Tang et al., 2020). Most
of the qualitative and quantitative HPLC and HPLC-MS methods have
been reported which are made up of modification of previously reported
analytical methods using C18 column and some time C8 also used with
different buffer systems as a mobile phase. All HPLC-MS methods have
been reported in positive ionization mode for MIAs (Renaudin 1984,
13
Journal of Ethnopharmacology 284 (2022) 114647
1985; Naaranlahti et al., 1987; Sim et al., 1994; Chu et al., 1997; Uniyal
et al., 2001; Zhao et al., 2001; Choi et al., 2002).
5.1. Analysis of monoterpene indole alkaloids
5.1.1. Qualitative analysis
Mostly of the qualitative analysis of MIAs has been reported on C18
column for separation with the mobile phase of a combination of
acetonitrile and methanol with a buffer like formic acid with isocratic or
gradient systems (Renaudin 1984, 1985; Bhadra and VaniShanks, 1993;
Moreno-Valenzuela et al., 1998; Rijhwani and Shanks, 1998; Lee and
Shuler, 2000; El-Sayed et al., 2004). A combination of different type of
S. Kumar et al.
Fig. 3B. Structures of monoterpene indole alkaloids (60–110) from C. roseus.
buffer solutions such as ammonium carbonate, ammonium acetate,
ammonium formate and sodium phosphate buffer have been also used
adequately as mobile phase for separation of MIAs by the many different
research groups (Auriola et al., 1989; Toivonen et al., 1991; Schröder
et al., 1999; Singh et al., 2000; Barthe et al., 2002; Tikhomiroff and
Jolicoeur, 2002; Batra et al., 2004; Lee-Parsons et al., 2004; Dutta et al.,
2005; Shams et al., 2009; Chen et al., 2013; Koel et al., 2020; Baig et al.,
2021). A different ratio of acetonitrile and methanol with a buffer like
14
Journal of Ethnopharmacology 284 (2022) 114647
formic acid was preferred for HPLC-MS analysis due to avoid suppres­
sion of ions during analysis while HPLC allow for all types of buffer
systems as mobile phase (Konermann, 2017 Susa et al., 2017). Many
research groups have identified and characterized compounds 27, 80,
16-O-demethylvindoline (34), and serpentine (95) in alkaloid extracts of
different parts of C. roseus using LC-MS (Chu et al., 1997; Choi et al.,
2002; Zhou et al., 2005; He et al., 2011; Chen et al., 2013; Kumar et al.,
2018a, 2018b; Jeong et al., 2018). Similarly, compounds 27, 34, 95,
S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Fig. 4A. Structures of bisindole alkaloids (111–133) from C. roseus.

15
S. Kumar et al.
Fig. 4B. Structures of bisindole alkaloids (134–145) from C. roseus.
111, 112, and vindolidine (38), vindolinine (43) and 19-S-vindolinine
(isomer of vindolinine) have been identified and characterized from the
leaves of an alkaloids extract using high-performance liquid
chromatography-electrospray ionization tandem mass spectrometry
(HPLC-ESI-MS/MS) (Rischer et al., 2006; Chen et al., 2011; Chen et al.,
2013; El-Domyati et al., 2014; El-Domyati et al., 2014; Ayob et al.,
2017). A total of 16 alkaloids have been identified and characterized in
extracts of whole stem tissue by HPLC-ESI-MS/MS (Luo et al., 2014;
Yamamoto et al., 2016). The HPLC-ESI-MS/MS method has been
developed for the characterization of compounds 27, 34, 111, 112 and
131 by comparison with the standards in three batches of C. roseus crude
samples collected from different regions of China. A capillary electro­
phoresis–mass spectrometry (CE-MS) method has been used for the
identification of compounds 111 and its monomeric precursors ( 27) and
compound 34 from stems of C. roseus (Chen et al., 2011). Compounds
27, 34, 80, 111 and 131 were identified from stems of C. roseus using
direct-injection in ion trap-mass spectrometry (IT-MS) (Chen et al.,
2013).
The ultra-performance liquid chromatography-quadrupole time-of-
flight (UPLCQ-TOF) mass spectrometry method has been developed for
the identification and characterization of compounds 27, 34, 80, 95,
111 and 112. A total of 22 indole alkaloids, including, compounds 27,
34, 80, 95, 55, 111 and 112 have been unambiguously characterized,
whereas around 16 compounds were tentatively identified (Jeong and
Lim, 2018). Ferreres et al. (2010) have been reported an
HPLC-ESI-MS/MS for identification and characterization of compounds
43, 34 and 80 along with isomers of compounds 43, 80 and 95 in
16
Journal of Ethnopharmacology 284 (2022) 114647
aqueous extracts of C. roseus.Thermo Betasil C8 column (250 × 4.5 mm,
5 μm) at 25 ◦ C used for chromatographic separation of MIAs using
mobile phase, 0.1% formic acid/water, and acetonitrile using
high-performance liquid chromatography (HPLC) coupled with elec­
trospray ionization quadrupole time of flight tandem mass spectrometry
(HPLC-ESI-QTOF-MS/MS) in positive mode (Kumar et al., 2018a).
Compounds 27, 52, 34, 111, 112 and 121 have been unambiguously
identified and characterized using diagnostic fragmentation pathways
(Kumar et al., 2018a). A total of 72 MIAs alkaloids which included 11
MIAs were unambiguously identified and characterized by comparison
with standards, whereas the remaining 62 MIAs were tentatively iden­
tified and characterized in ethanolic extracts of leaves, stem and roots of
the plant. The method has been very useful for the separation and
identification of isomers of well known compounds. Distribution of all
the MIAs have been investigated in 30 samples of C. roseus collected
from five states of India. The approach considered as rapidly and
effectively characterizes the targeted and untargeted MIAs in C. roseus
using developed analytical methods together with diagnostic fragment
patterns (Kumar et al., 2018a). Total 172 alkaloids mass spectrometric
data for dereplication along with computer-based approaches, such as
molecular networking has been reported which are the maximum
number of MIAs identified in this plant till date (Ramos et al., 2019).
Relatively advanced approach 13carbon (13C) and 15nitrogen (N) label­
ing along molecular networkinghas with applied on HPLC-MS/MS data
by Nakabayashi et al. (2020). The characteristics of two types of skel­
eton namely indole basic and oxindole skeletons have been have pro­
posed on the basis of mass (m/z) shifting of the product and precursor
S. Kumar et al.
Fig. 5A. Structures of flavonoids (146–166) from C. roseus.
ions. Total of 86 and 73 common monoisotopic ions of indole basic and
oxindole skeletons reported, respectively. The comparative analyses of
the three pairs of stereoisomers showed more than 170 common mon­
oisotopic ions in each pair (Nakabayashi et al., 2015). Finally, the mo­
lecular formula of 45 MIAs was unambiguously identified using isotopic
15
N. The alkaloid network analysis showed the structural commonality
and uniqueness among the MIAs. Some of them, antirhine was identified
using an authentic standard in leaves of this plant (Nakabayashi et al.,
2020). Serpentinine-related bisindole alkaloids have been characterized
using using LC-MS/MS and NMR guided by a molecular
networking-based dereplication strategy in ethanolic extracts Picralima
nitida (Alcover et al., 2020). The investigation of published literature
arrive at a conclusion that the MS/MS analysis with isotopic labelling
along with computer based networking analysis provides the confidence
of dereplication MIAs in complex metrix.
5.1.2. Quantitative analysis
The large-scale quantification of compounds 111 and 112 from
different extracts of plant parts of C. roseus made special attention to
17
Journal of Ethnopharmacology 284 (2022) 114647
pharmaceutical industries. Like qualitative analysis, numerous methods
have been reported for the quantitation of compounds 111 and 112
along with different alkaloids using both C18 and C8 column with buffer
system as mobile phase (BlaskÓ and Cordell, 1990; Favretto et al.,
2001). These compounds have been quantified from ethanolic extract
using TLC (Tam et al., 1995). Compounds 111 and 112 have been
typically quantified using TLC and HPTLC. Similarly, quantitation of
compounds 111 and 112 has been reported by TLC and HPLC and
ultraviolet–visible (UV–Vis), HPLC-ESI-MS, and HPLC-ESI-MS/MS
(Toivonen et al., 1991; Zhang et al., 2017). Another bisindole alka­
loid, isoleurosine has been quantified using UV and TLC (Mon­
forte-González et al., 1992; Nagy-Turak and Vegh, 1994; Parthasarathy
et al., 2020). Similarly, detection of various bisindole alkaloids from
extracts of C. roseus and its cell culture have been reported by HPLC
coupled with different detectors such as photo diode array (PDA),
fluorescence and mass spectrometry (MS) for quantitative analysis
(Wittkamp et al., 1997; Gupta et al., 2005; Kumar et al., 2013;
Thompson, 2017; Thiäner et al., 2019).
Several improvements and developments were made to make HPLC
S. Kumar et al.
Fig. 5B. Structures of pronthocynine (167–179) and phenolic acids (180–188) from C. roseus.
an even more powerful analytical technique and enhance the capacity
via integrated more powerful detectors such as mass spectrometry (MS).
The measurement of molecular weight for each peak is considered
highly useful to analyze plant or cell extracts by liquid chromatogra­
phy–mass spectrometry (LC-MS). A method for quantitative analysis of
MIAs in plant extracts has been reported by Van der Heijden et al. (1987)
using mass spectrometry for the first time. The HPLC coupled with
electrospray ionization (ESI) ion source enhanced the detection of
compounds 112 and 111 in plant extracts at a limit of detection of 0.2
μg/mL. The sensitivity for MIAs was 2–3 times higher in ESI-MS rather
than with UV detection (Chu et al., 1997; Zhao et al., 1999). Most of the
quantitative HPLC and LC-MS methods have been validated according to
the ICH guidelines.
Due to the advancement of analytical instruments, compounds 111
and 131 have been identified in C. roseus by direct infusion into ESI
18
Journal of Ethnopharmacology 284 (2022) 114647
source using an IT-MS instrument in the positive ion mode (Zhou et al.,
2005; Kumar et al., 2018b). These HPLC-MS/MS methods showed good
signals corresponding to the protonated molecular ions [M+H]+ and
product ions (Zhang et al., 2017). The MS combined with ESI provided a
high selectivity and sensitivity for the determination of MIAs in positive
mode. The separation, identification and quantification of MIAs after
validation of the developed methods were applied in extracts of C. roseus
using HPLC with UV and MS detection. The amount of compounds 111
and 131 observed were 1.8, 2.10, 6.02 and 7.52 μg/g by LC–UV and
LC-MS, respectively (Chen et al., 2013). Compounds 111, 112 and 131
were separated on a C18 HPLC column with the mobile phase of meth­
anol with buffer 15 nmol/L ammonium acetate with 0.02% formic acid
using multiple reaction monitoring (MRM) mode. This method was
validated and the results observed as the intra- and inter-day precision
and accuracy of these alkaloids were within 1.2–11.5 (RSD: relative
S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Fig. 6A. Structures of volatile compounds (alkanes (189–203), alkenes (204–220), aldehydes (221–247), ketones (248–270) from C. roseus.

19
S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Fig. 6B. Structures of volatile compounds (alcohols (271–303), acids (304–318), esters (319–331), acetates (332–337), lactones (338–344) from C. roseus.

20
 S. Kumar et al.
Table 4
Separation and analytical techniques and their conditions used for analysis of Catharanthus alkaloids in different extracts.
S.N. Instrument Analytes Column Mobile Phase Detection Elution Range of RT Flow Plant part Extract Reference
program (min) rate
(mL/
min)

1. HPLC Ajmalicine, μBondapack C18 Methanol:5 mM (NH4)2HPO4 (pH UV254 Isocratic 12.00–19.00 1 Cell Ethanolic Renaudin (1984)
catharanthine, (300 × 3.9 mm, 7.3) (71:29, v:v) suspension
serpentine and 10 μm) (Waters) cultures
vinblastine
Ajmalicine, μBondapack C18 Methanol:5 mM (NH4)2HPO4 (pH UV254 Isocratic 9.5–22 1 Hairy roots, Aqueous solution Renaudin (1985)
catharanthine, (300 × 3.9 mm, 7.3) (67:33, v:v) cell with 001% acetic
tetrahydroalstonine 10 μm) (Waters) suspensions acid
and vindoline
Ajmalicine, Spheri-5 C18 (220 Solvent A: 0.2% TEA in 25 mM PDA Gradient 13–24.5 Cell Ethanolic Naaranlahti et al.
catharanthine, × 4.6 mm, 5 μm) CH3COONH4; Solvent B: 0.2% TEA suspension (1987)
serpentine, in a mix of methanol: acetonitrile cultures
vinblastine, (29:71, v:v)
vincristine and
vindoline
Ajmalicine, μBondapak C18 Acetonitrile: 0.1 M CH3COONH4 UV280 Gradient 2.0–18.0 1 Tissue Ethanolic Auriola et al. (1989)
catharanthine, (300 × 3.9 mm, (pH 7.2) (49:51, v:v) cultures
serpentine, 10 μm)
tabersonine and
tryptamine
Catharanthine and Supelcosil LC-18- 0.1 M sodium acetate: methanol: UV 0–20 1 Catharanthus ethanol Naaranlahti et al.
ajmalicine DB (150 × 4.6 acetonitrile (49: 15: 36, pH 6.5) roseus cell (1989)
mm, 5 μm) culture
21

Ajmalicine, Hypersil ODS C18 Solvent A: 14 mM TEA in PDA Gradient N/A 0.4 Hairy root Ethanolic Toivonen et al.
catharanthine and (100 × 2.1 mm, 5 CH3COONH4; Solvent B: 14 mM cultures (1991)
tabersonine μm) (Agilent) TEA in Methanol: acetonitrile
(50:50, v:v)
Ajmalicine, μBondapack C18 Methanol:5 mM (NH4)2HPO4 (pH UV254 Isocratic 8.5–59.20 0.8 Hairy roots, alkaloidal Bhadra and
catharanthine, (300 × 3.9 mm, not mentioned) (67:33, v:v) cell VaniShanks (1993)
serpentine, 10 μm) (Waters) suspensions
vinblastine and
vindoline
Catharanthine, Hypersil ODS Solvent A: Water, 0.1% TEA; PDA Gradient N/A Cell Ethanolic Aerts et al. (1994)
tabersonine and (200 × 2.1 mm, 5 Solvent B: Acetonitrile, 0.1% TEA suspension
vindoline μm) (Agilent) cultures
Ajmalicine and μBondapack C18 Methanol: acetonitrile: 5 mM UV254 Isocratic NA 1 Cell alkaloidal Sim et al. (1994)
catharanthine (300 × 3.9 mm, (NH4)2HPO4(pH 7.3) (30:40:30, v: suspension
10 μm) (Waters) v:v) cultures

Journal of Ethnopharmacology 284 (2022) 114647

16- Nova-Pak C18 Methanol: water: TEA (75:25:0.1, v: PDA Gradient 30.5 0.6 Cell Crude protein St-Pierre and De
methoxytabersonine (300 × 3.9 mm, 4 v:v) suspension Luca (1995)
μm) (Millipore) cultures
Ajmalicine and Ultrasphere ODS Acetonitrile:10 mM (NH4)2HPO4, UV280 Isocratic 5.9–21.80 1.5 Hairy roots, Not reported Moreno-Valenzuela
yohimbine C18 (250 × 4.6 (43:57, v:v) cell et al. (1998)
mm, 4 μm) suspensions
(Beckman)
Ajmalicine, Bondclone C18 Methanol: acetonitrile: 5 mM PDA Isocratic NA 1 Cell alkaloidal Rijhwani and
hörhammericine, (300 × 3.9 mm, (NH4)2HPO4 (32:32:36, v:v:v) suspension Shanks (1998)
lochnericine, 10 μm) cultures
serpentine and (Phenomenex)
tabersonine
16- Solvent A:0.2% NaOH in water, PDA Gradient 13.0–26.7 1 Schröder al.et.
hydroxytabersonine, Solvent B: Acetonitrile (1999)
(continued on next page)
 Table 4 (continued )

S. Kumar et al.
S.N. Instrument Analytes Column Mobile Phase Detection Elution Range of RT Flow Plant part Extract Reference
program (min) rate
(mL/
min)

16-methoxytaberso­ Nucleosil C18

nine and tabersonine (200 × 4 mm, 5
μm) (Agilent)
Catharanthine, μBondapack C18 Acetonitrile:0.1 M Phosphate PDA Isocratic 10.15–13.54, 0.6 Leaves 90% ethanolic Singh et al. (2000)
vinblastine, vindoline (300 × 3.9 mm, buffer: glacial acetic acid (pH 4.14)
and vincristine 10 μm) (Waters) (38:62:0.3, v:v:v)
Catharanthine, Symmetry C18 Solvent A (pH 7.0): Methanol: PDA Gradient 14.62–19.6 Cell – Uniyal et al.,(2001)
vinblastine (150 × 4 mm, 5 acetonitrile:5 mM CH3COONH4: suspension
Vincristine and μm) (Waters) triethylamine (13:32:55:0.2, by cultures and
vindoline volume); Solvent B (pH 7.0): plant parts
Methanol: acetonitrile:5 mM
CH3COONH4: triethylamine
(19:46:35:0.2, by volume)
Ajmalicine, Nucleosil 5 C18 Methanol: acetonitrile: 25 mM PDA Gradient N/A 1 Cell Methanolic Zhao et al. (2001)
catharanthine and (250 × 4.6 mm, 5 CH3COONH4: TEA (15:40:45:0.1, suspension
serpentine μm) (Agilent) by volume) cultures
Anhydrovinblastine, XTerra C18 (250 Acetonitrile: water: boric acid (pH UV214 Isocratic 6.0–20.8 1 – – Barthe et al. (2002)
catharanthine, × 4.6 mm, 5 μm) 10) (55:45:3.1, v: v: w) nm
vindoline, vinflunine (Waters)
and viorelbine
Catharanthine, Zorbax XDB C18 Solvent A: 5 mM KH2PO4 (pH 6); UV Gradient 7.8–20.8 2 Hairy root Tikhomiroff and
serpentine, (250 × 4.6 mm, 5 Solvent B: Acetonitrile cultures Jolicoeur (2002)
tabersonine, μm) (Agilent)
vinblastine, vindoline
and vincristine
22

Ajmalicine and μBondapack C18 Acetonitrile:0.1 M Phosphate UV Isocratic 0.6 Root 90% ethanolic Batra et al. (2004)
serpentine (300 × 3.9 mm, buffer:glacial acetic acid (pH 4.14)
10 μm) (Waters) (38:62:0.3, v:v:v)
Ajmalicine, Phenomenex DAD NA Leaf and root Digvijay et al.
catharanthine, Luna C18 (250 × (2004)
loganine, serpentine, 4.6 mm, 5 μm)
tryptamine,
tryptophan,
vinblastine,
vincristine and
vindoline
Ajmalicine, C18 (125 × 4 mm, 0.025 M UV233, Gradient – 1 Leaves, stems, Methanolic Favali et al. (2004)
serpentine, 5 μm) methanol–acetonitrile–ammonium 254, 280 and roots
vinblastine and acetate

Journal of Ethnopharmacology 284 (2022) 114647

vindoline
Catharanthine, Vydac C18 (250 × Acetonitrile:0.1% (w:v) TFA in UV254 Isocratic 7.2–23.5 1 Cell alkaloidal El-Sayed et al.
stemmadenine and 4.6 mm, 5 μm) water (21:79, v:v) suspension (2004)
tabersonine (Vydac) cultures
Ajmalicine, μBondapack C18 Acetonitrile: 0.1% (w:v) UV254 Isocratic 19.67–23.14 1 Cell – Lee-Parsons et al.
catharanthine, (300 × 3.9 mm trifluoroacetic acid (TFA) in water suspension (2004)
serpentine 10 μm) (Waters) (22:78, v:v) cultures
Ajmalicine, Phenomenex 12% acetonitrile: 88% water + UV254 Isocratic May-37 1 Cell Methanolic Wong et al. (2004)
serpentine and Luna C18 (150 × 0.1% formic acid suspension
tryptamine 4.6 mm, 5 μm) cultures
Ajmalicine, Waters’ıBondapk CH3OH: 5 mM NH4H2PO4(7: 3, v/v, UV – 1 Suspension Methanolic Zheng &Wu (2004)
tryptamine and C18 (250 × 4.6 pH 7.1) culture of the
tryptophan mm, 5 μm) cell line
UV Gradient 2 Leaves 90% ethanolic Dutta et al. (2005)
(continued on next page)
 S. Kumar et al.
Table 4 (continued )
S.N. Instrument Analytes Column Mobile Phase Detection Elution Range of RT Flow Plant part Extract Reference
program (min) rate
(mL/
min)

Catharanthine, Zorbax XDB C18 Solvent A: 5 mM KH2PO4 (pH 6);

serpentine, (250 × 4.6 mm, 5 Solvent B: Acetonitrile
tabersonine, μm (Agilent)
vindoline, vincristine
and vinblastine
Catharanthine, Chromolith 21:79 (v/v) acetonitrile–0.1M UV254 Gradient 6.81–26.93 1.2 Leaves 90% Ethanolic Gupta et al. (2005)
vinblastine, Performance C18 phosphate buffer containing 0.5%
Vincristine and (100 × 4.6 mm) glacial acetic acid
vindoline
Ajmalicine, Luna C18 (150 × Solvent A: Water, Solvent B: UV Gradient 5.0–37.0 1 Hairy root Lee-Parsons et al.
serpentine and 4.6 mm 5 μm) Acetonitrile, Solvent C: Formic acid cultures (2004)
tryptamine (Phenomenex) (kept at 0.1% of the total elution
volume)
Catharanthine, Inertsil ODS-3 C18 5 mM sodium phosphate pH 6.0 UV220,230 Gradient 2 Leaves alkaloidal Murata and Luca
tabersonine and (4 × 250 mm) (solvent A) and acetonitrile (solvent (2005)
vindoline B)
Vinblastine, Agilent Zorbax 0.1% Triethylamine (solvent A) and UV280 Gradient 10.0–40.0 0.8 Leaves Commercial Zhou et al. (2005)
vincristine, vindoline Eclipse XDB-C8 methanol (solvent B)
and vindolidine (150 × 4.6 mm, 5
μm)
Ajmalicine, Phenomenex 50% MeOH/50% MeCN: 5 mM UV218 Gradient 1 Hairy roots Methanolic and Peebles et al. (2006)
catharanthine, Luna 5A C18 (250 (NH4)2HPO4 (pH 7.3) alkaloidal
horhammericine, × 4.6 mm)
lochnericine, (Torrance, CA)
23

serpentine,
tabersonine,
tryptamine and
tryptophan
Catharanthine, ODS C18 (250 × water + 0.1% Formic acid and B UV220 – Leaves and Methanolic Guo et al. (2007)
vinblastine, vindoline 4.6 mm) (Waters) acetonitrile + 0.1% formic acid roots
and vincristine
Horhammericine BEH C18 (1.0 × A: methanol: acetonitrile:5 mM: UV Isocratic – Hairy root alkaloidal Magnotta et al.
50 mm 1.7 μm NH4OAc:Et3N (5.8:14.2:80:0.2 by cultures (2007)
mm) (Waters, volume) B: MeOH:MeCN:5 mM:
Milford, MA) NH4OA-c:Et3N(23:57:20:0.2 by
volume), C: MeOH: MeCN:5 mM:
NH4OAc:Et3N(26:64:10:0.2 by
volume)

Journal of Ethnopharmacology 284 (2022) 114647

Vinblastine HypersilBDS-C18 5 mM Na2HPO4 x 2H2O buffer (pH UV218 Gradient 1.5 Leaves alkaloidal Verma et al. (2007)
(150 × 4.6 mm, 5 adjusted to 7.3 by H3PO4)-
μm) (Agilent) acetonitrile-methanol
Ajmalicine, Bondpak C18 Acetonitrile and ammonium UV254 Gradient 20.52–41.42 Leaves, Root- Ataei-Azimi et al.
cataranthine, (1500 × 2.5 mm, carbonate buffer pH 7.3 (1:1) callus and (2008)
vinblastine, 4.6 μm) roots
Serpentine,
vincristine and
vindoline
Catharanthine and Nucleosil 5 C18 50 mM sodium phosphate pH 3.9: UV280 Leaves Dichloromethane Ramani and
vindoline (250 × 4.6 mm, 5 acetonitrile: 2 methoxyethanol Suspension Jayabaskaran
μm) (Novapak) (80:15:5 v/v)] cultures (2008)
Ajamalicine, 0.01M phosphate buffer (pH 4.9) UV220 Gradient C. Roseus/ 90% ethanolic Singh et al. (2008)
catharanthine, and acetonitrile root, stem,
(continued on next page)
 S. Kumar et al.
Table 4 (continued )
S.N. Instrument Analytes Column Mobile Phase Detection Elution Range of RT Flow Plant part Extract Reference
program (min) rate
(mL/
min)

serpentine, Phenomenex C18 leaf, flower

vincristine, (250 × 4.6 mm, and seed
vinblastine and 5¼ m)
vindoline
Catharanthine, Phenomenex 10 mM ammonium acetate buffer UV220 Gradient 0.3 Leaves methanolic Verma et al. (2008)
vinblastine, Gemini C18 (150 (pH 5.0), acetonitrile and methanol
vincristine and × 2.0 mm, 5 μm)
vindoline (Torrance, CA,
USA)
Vincristine Merck LiChro Acetonitrile: methanol; 3:1 UV220 Isocratic 1 Leaf, root and alkaloidal Aslam et al. (2009)
CART C18 (125 × whole Plant)
4.6 mm, 5 μm)
Vincristine Supelco ODS Methanol:Water (50:50 v/v) UV280 Isocratic 0.5 Leaf, stem Methanolic Kalidass et al.
Hypersil, C18 (4.6 (2009)
× 250 mm, 5 μm)
Vinblastine and Nova pack C18 Phosphate buffer solution of pH 6.5, UV Isocratic 8.05–11.83 6 C. Roseus Methanolic, Shams et al. (2009)
vincristine (3.9 × 150 mm) acetonitril (55 : 45) Alkaloidal
(Waters)
А-3′ ,4′ - Waters ODS C18 A: water∶ diethylamine, pH to 7.2 UV220 Gradient – 1.5 Leaves Methanolic Tang et al. (2009)
anhydrovinblastine, (250 × 94.6 mm, H3PO4, B: methanol∶ acetonitrile =
catharanthine and 5 μm) 4∶1
vinblastine, vindoline
Vinblastine Microsorb-MV Methanol and acetonitrile UV240 Gradient 7.02 0.2 Leaves Aqueous Ahmed et al.
C18 (150 × 4.6 (2010b)
24

mm, 5 μm)
Vinblastine Merck LiChro Acetonitrile and methanol, 3:1 UV255 – 1 Leaves/leaf Methanolic Aslam et al. (2010)
CART C18 (125 × callus and in
4 mm, 5 μm) germinating
embryos
Catharanthine, ODS C18 (250 × 4 Water (containing 0.005mol.L-1 UV280 Gradient 7.52–31.36 1 Leaves alkaloidal Tang and Rao
tryptamine and mm) Agilen ammonium hydrogen phosphate), (2010)
vindolin Hypersil, methanol (containing 0.67%
California, USA) triethylamine), and acetonitrile
Ajmalicine, Phenomenex 50% Methanol/50% Acetonitrile: 5 UV218 – 1 Hairy root Methanolic Li et al. (2011)
catharanthine, Luna 5A C18 (250 mM (NH4)2HPO4 (pH 7.3) cultures
lochnericine, × 4.6 mm)
serpentine and (Torrance, CA)
tabersonine

Journal of Ethnopharmacology 284 (2022) 114647

Catharanthine, Zorbax Eclipse Methanol:acetonitrile:ammonium UV Isocratic 10.26–19.62 1 Leaves, stem, Methanol and Siddiqui et al.
vinblastine, vindoline plus C18 (250 × acetate buffer (25 mM) with 0.1% and flower methanol: water (2011)
and vincristine 4.6 mm, 5 μm) triethylamine (15:45:40 v/v)
Catharanthine, HiQ sil-C18 (4.6 × Methanol: acetonitrile (4:1, v/v, UV220 – 10.0–26.0 1.5 Leaves 85% ethanolic Yang et al. (2011)
vinblastine and 250 mm, 5 μm) solution A) water:diethylamine
vindoline, (KYA TECH) (986:14, v/v) with phosphoric acid
pH of 7.5 (solution B).
Catharanthine, HiQ sil C18 (4.6 × A (methanol/acetonitrile), and UV220 Gradient 9.0, 11.9, 2 Leaves 80% ethanolic Mu et al. (2012)
vinblastine, 250 mm) solution B (water/diethylamine, pH 13.2, 20.2
vincristine and 7.2 by H3PO4)
vindoline
β-carotene C18 (250 × 4 mm, Methanol (A), water/methanol UV240 Gradient 0.5 Cell Acetone Thabet et al. (2012)
3 μm) (20:80, v/v) containing 0.2% suspensions
leaves
(continued on next page)
 S. Kumar et al.
Table 4 (continued )
S.N. Instrument Analytes Column Mobile Phase Detection Elution Range of RT Flow Plant part Extract Reference
program (min) rate
(mL/
min)

Langerwehe, ammonium acetate (B) and

Germany) tertmethyl butyl ether (C)
Ajmalicine, C18 (Acquity, 98.9% Deionised water, 1.0% UV Isocratic 1 Leaves Homogenised Andrade et al.
catharanthine, Waters, acetonitrile and 0.1% formic acid with acid (2013)
serpentine, Manchester, UK) water+0.01%
vinblastine vindoline formic acid) 1:1
and vincristine (m/v)
Ajmalicine, Mediterranean Methanol-acetonitrile-ammonium MS Gradient 1 leaves Alkaloidal Fernández-Pérez
catharanthine, sea C18 (150 × acetate (0.0025 mol/L) and et al. (2013)
lochnericine and 4.60 mm, 3 μm triethylamine (0.0072 mol/L)
tabersonine Teknokroma, (10:40:50:0.1)
Spain
Vinblastine and C18 (Waters) 5%–95% Acetonitrile in water with UV254,220 Gradient 0.5 Endophytic Alkaloidal Kumar et al. (2013)
vincristine 0.01% trifluoroacetic acid fungus
Fusarium
oxysporum
Catharanthine, himpak VP- Methanol, acetonitrile, and 5 mm UV220 Isocratic 1 Suspension Methanolic Pandiangan et al.
tryptamine, ODSC18 (150 × diammonium hydrogen phosphate culture (2013)
vinblastine and 4.6 mm) (3:4:3)
vindoline
Ajmalicine C18 (4.6 × 150 UV220 Gradient Root Methanolic Thakore et al.
mm 5 μm) (2013)
(Agilent
Technologies)
25

Tryptophan C18 (100 × 4.6 21:79 (v/v) acetonitrile: 0.1 M UV254 Gradient 1.2 Cell alkaloidal Verma et al. (2013)
mm) phosphate + 0.5% glacial acetic suspensions
acid of
Catharanthine and Capcell pak C18 Acetonitrile 20 mM sodium- UV250 Gradient 1 Leaves Methanolic Fukuyama et al.
vindoline (Shiseido Co., phosphate buffer (pH 7.0) (2015)
Ltd., Japan) acetonitrile
Anhydravinblastine, Kromasil C18 Water: diethylamine (986:14, v/v) UV220 2 Leaves 80% Ethanolic Luo et al. (2014)
catharanthine, (250 × 4.6 mm, 5 with phosphoric acid to a pH of 7.5
vinblastine, μm) (solution A) and methanol:
vincristine and acetonitrile (4:1,v/v, solution B).
vindoline
Vinblastine and Octadecylsilane Acetonitrile: Water 50:50 UV Isocratic 1.229–1.474 2 Leaves Methanolic Saravanan and
vincristine C18 (250 × 4.6 Karthi (2014)
mm, 5 μ (Supelco)

Journal of Ethnopharmacology 284 (2022) 114647

Ajmalicine C18 (4.6 × 150 0.01 M phosphate buffer (pH 4.9) UV220, Gradient 11 1.8 Hairy roots alkaloidal Thakore et al.
mm, 5 μm and acetonitrile 254 (2015)
(Agilent
Technologies
1200 series, USA)
Ajmalicine, Phenomenex Methanol/acetonitrile/10 mM UV280 – 1 Suspension- 75% ethanolic Zhou et al. (2015)
catharanthine and Gemini C18 (250 ammonium acetate (15:40:45,v/v/ cultured cells
vindoline × 4.6 mm, 5 μm) v)
(Phenomenex
Inc., Torrance,
CA, USA)
Catharanthine, C18 (250 × 4.6 0.025M ammonium acetate UV220 Gradient 9.00–33.00 1 Leaves Methanolic Zhu et al. (2015)
ajmalicine, mm) Waters solution and acetonitrile
(continued on next page)
 S. Kumar et al.
Table 4 (continued )
S.N. Instrument Analytes Column Mobile Phase Detection Elution Range of RT Flow Plant part Extract Reference
program (min) rate
(mL/
min)

strictosidine and
vindoline
Ajmalicine and C18 (150 × 2.1 A: 0.1% formic acid in water, B: UV254 Gradient 12.34–13.09 0.2 Hairy root Methanolic Benyammi et al.
catharanthine mm, 5 μm) 0.1% formic acid in methanol MS lines (2016)
Alltima, Alltech,
Saint Jean de
Gonville, France)
Vinblastine, C18 (250 × 4 mm, 55% sodium phosphate buffer (5 UV Gradient NA 0.7 Root Methanolic Hanafy et al. (2016)
vincristine and 5 μm) mmol/l, pH 6), 45% acetonitrile)
catharanthine
Vindoline C18 (250 × 4.6 70:30 (v/v) mixture of 100 mM MS Gradient 1 Leaves tissues Methanolic Pandey et al. (2016)
mm, 5 μm) ammonium acetate (pH 7.3) (A):
(Waters) acetonitrile (B)
Vinblastine Agilent Zorbax Ammonium acetate (20 mM) 70:30 UV254 Gradient 0.2 Leaves Carbon dioxide Falcão et al. (2017)
Eclipse XDB-C18 acetonitrile, at 30 and ethanol
Ajmalicine, Diamonsil C18 water: diethylamide(v/v), pH 7.2], UV220 Gradient NA 1.5 Leaf, root Methanolic Hashemabadi et al.
catharanthine, (250 × 4.6 mm, 5 methanol: acetonitrile = 4:1 (v/v) (2018)
vinblastine, μm)
vincristine and
vindoline
Ajmalicine, C18 (50 × 2.1 mm, Water (solvent A) and acetonitrile UV Gradient 5.47–11.25 0.4 Root, stem, 70% Methanolic Jeong and Lim
catharanthine, 1.7 μm) (solvent B) containing 0.1% formic and leaf (2018)
serpentine, acid ammonium formate
vinblastine,
26

vincristine and
vindoline
Catharanthine, Eclipse XDB-C18 A and TFA-water-acetonitrile UV254 Gradient 1 CMC Methanolic Moon et al. (2018)
vinblastine, (4.6 × 250 mm, 5 (0.01:5:95) as solvent B suspension
vincristine and μm) (Agilent culture
vindoline Technologies)
Vinblastine Microsorb - MV Methanol – phosphate buffer (5 UV254 Gradient 20 2 Leaves Aqueous Rahim et al. (2018)
C18 (250 × 4.6 mM, pH 6.0) – acetonitrile
mm, 5 μm)
Vincristine Zorbax SB-C18 Methanol-15 mmol⋅L− 1 UV254 1 Aerial parts Carbon dioxide Karimi and Raofie
(2.1 × 50 mm, ammonium acetate containing and ethanol (2019)
1.8 μm) 0.02% formic acid (65: 35, V/V)
Kaempferol, Athena C18 (250 Acetonitrile, Phosphate buffer (pH UV254 Gradient 2.42–9.46 1 Leaves Ethanolic Rao and Ahmed
quercetin and rutin × 4.6 mm) = 5.8) powders (2014)

Journal of Ethnopharmacology 284 (2022) 114647

Gallic acid, quercetin hypersil gold C18 Acetonitrile, methanol, and 3% UV254 Gradient Root, stem, Deep eutectic Nisar (2017a);
and rutin (250 × 4.6 mm, 5 formic acid (50/50/0.3, v/v/v) leaves, and solvent Nisar (2017b)
μ) flower petal
7,3-O- Phenomenex 3% Aqueous formic acid (A) and UV520 Gradient 0–40 min 1 Flower petals Natural deep Dai et al. (2016)
dimethylcyanidin, 3- Luna C18 (4.6 × methanol with 3% formic acid eutectic solvents
O-(6-O-p-coumaroyl) 250 mm, 5 μm)
glucose, hirsutidin 3-
O-(6-O-p-coumaroyl)
glucose, 7-O-
methylcyanidin,
malvidin 3-O-(6-O-p-
coumaroyl) glucose
and petunidin
2. UPLC UV280 Isocratic 1 Leaves 75% ethanolic Zhou et al. (2015)
(continued on next page)
 S. Kumar et al.
Table 4 (continued )
S.N. Instrument Analytes Column Mobile Phase Detection Elution Range of RT Flow Plant part Extract Reference
program (min) rate
(mL/
min)

Ajmalicine, Phenomenex Methanol/acetonitrile/10 mM

catharanthine and Gemini C18 (250 ammonium acetate (15:40:45, v/v/
vindoline × 4.6 mm, 5 μm) v)
3. HPLC-MS Catharanthine, Hypersil ODS Solvent A: 25 mM CH3COONH4 in MS Gradient 14.8–21.3 1 Leaf Isopropyl Chu et al. (1997)
vinblastine, (200 × 4.6 mm, 5 methanol CH3COONH4 in water; alcoholic
vincristine, μm) (Agilent) Solvent B: 25 mM
vindoline,
vindolidine,
vindolinine and 19S-
vindolinine
Vinblastine and Zorbax Bonus C18 Solvent A: 20 mM CH3COONH4; MS Gradient 18–21.5 0.2 Aerial parts Methanolic Choi et al. (2002)
vincristine (150 × 2.1 mm, 5 Solvent B: Acetonitrile and roots
μm) (Agilent)
Vinblastine, Direct flow Methanol MS N/A – Whole plant Commercial Zhou et al. (2005)
vincristine, vindoline injection
and vindolidine
Vinblastine and Xterra MS C18 10 mM ammonium acetate (pH 10) MS Isocratic 1 Leaves Ethanolic Rischer et al. (2006)
vincristine (4.6 × 150 mm, 5 and acetonitrile (55:45) Suspension
μm) (Waters) cultures
Vincristine, Phenomenex Solvent A (46%) + 0.1% TFA/ MS Isocratic 5–14.40 0.2 Plasma Schmidt et al.
vinblastine and Polar-RP (250 × water; solvent B (54%) + 1:1 (2006)
vinorelbine 2.0 mm, 4 μm) methanol: acetonitrile.
Ajmalicine, Luna C18 (250 × acetonitrile (A) and acetic acid 1% MS Gradient 10.0–40.0 1 Root Methanol: water Ferreres et al.
ajmalicine (isomer), 4.6 mm, 5 μm) (B) (1:1) (2010)
27

catharanthine, (Phenomenex,
serpentine, United Kingdom)
serpentine (isomer),
tabersonine,
vindolinine and 19-S-
vindolinine
Vinblastine Merck LiChro Acetonitrile: methanol (3:1) UV255 Gradient 1 Embryos/ Chloroform Aslam et al. (2011)
CART C18 (125 × tissues
94 mm, 5 μm)
Ajmalicine, Waters Acquity Acetonitrile (A) and 0.1% formic MS Gradient 2.08–3.03 0.3 Cell line 80% ethanolic He et al. (2011)
catharanthine, UPLC®BEH C18 acid in water (B). c20hi
serpentine and (50 × 2.1 mm,
vindoline 1.7 μm)
Vindoline Acquity Ultra 50∶50 Acetonitrile-formic acid in MS Isocratic 0.5 Leaves Methanolic Liscombe &
BEH C18 (100 × water alkaloidal O’Connor (2011)

Journal of Ethnopharmacology 284 (2022) 114647

2.1 mm, 1.7 μm)
Ajmalicine, C18 (Acquity, 98.9% Deionised water, 1.0% MS Isocratic 1 Leaves Homogenised Andrade et al.
catharanthine, Waters, acetonitrile and 0.1% formic acid with acid (2013)
serpentine, Manchester, UK) water+0.01%
vinblastine, formic acid) 1:1
vincristine and (m/v)
vindoline
Ajmalicine, DL-Cl8 (250 × 4.6 Acetonitrile (A) and 10 mmoL UV, MS Gradient 1–0.5 Leave Alkaloidal Chen et al. (2013)
catharanthine, mm, 5.0 μm) ammonium acetate in water (B)
vinblastine vindoline (Japan)
and vinleurosine
Catharanthine; BEH C18 (50 mm 0.1% formic acid (A) and MS Gradient 1.74–4.47, 0.5 Leaf Methanol: water El-Domyati et al.
serpentine, × 2.1 mm, 1.7 acetonitrile (B) (1:1) (2014)
taberosinine, μm)
(continued on next page)
 S. Kumar et al.
Table 4 (continued )
S.N. Instrument Analytes Column Mobile Phase Detection Elution Range of RT Flow Plant part Extract Reference
program (min) rate
(mL/
min)

vinblastine,
vincristine,
vindoline,
vindolidine,
vindolinine and 19-S-
vindolinine
Catharanthine, Agilent Eclipse Methanol mmol/L ammonium MS Gradient 2.51–2.97 1 Leaves, stems, Water− 0.1% Zhang et al. (2014)
vinblastine, C18 (150 × 4.6 acetate containing 0.02% formic (MRM) roots H2SO4 methanol
vincristine and mm, 5 μm) acid (65 : 35, V/V)
vindoline,
vinleurosine
Catharanthine and C18 (2.1 × 50 mm, Acetonitrile–0.1% formic acid in MS Gradient 0.4 Plasma Lin et al. (2015)
vindoline 3.5 μm) water
Ajmalicine, Xselect CSH solvent A: water with 0.1% formic MS Gradient 7.0–13.0 0.3 Roots 80% methanolic Nakabayashi et al.
catharanthine, Phenyl-Hexyl acid) and solvent B: (acetonitrile (2015)
hirsuteine, hirsutine, (2.1 × 150 mm, with 0.1% formic acid)
formosanine, 3.5 μm) Waters,
isoformosanine, Milford, MA,
isorhynchophylline, USA)
isopteropodine,
mitraphylline
pteropodine and
rhynchophylline
Catharanthine, Phenomenex methanol/acetonitrile/10 mM MS Isocratic 1 Root-callus 75% ethanolic Zhang et al. (2015)
28

vinblastine, Gemini C18 (250 ammonium acetate (15:40:45, v/v/ and roots
Vincristine and × 4.6 mm, 5 μm) v)
vindoline,
Catharanthine, Cosmosil C18 (4.6 (A) 0.05 mol/L ammonium acetate MS Gradient 4.88–36.37 1 Leaves and Water: methanol Liu et al. (2016)
serpentine, × 250 mm, 5 μm), and (B) acetonitrile, Acetonitrile/ Human serum (50:50, v/v) and
tabersonine, WondaSil C18 0.2% formic acid in water, 37:63, v/ samples alkaloidal
tryptamine, (200 × 4.6 mm, 5 v
vinblastine, μm)
vincamine and
vindoline and
yohimbine
16 alkaloids Eclipse XDB-C18 25 mM ammonium acetate and LTQ Isocratic 0.25 Stem tissue Yamamoto et al.
(150 × 4.6 mm, 5 acetonitrile Orbitrap at 20:80 (2016)
μm) (Agilent)
Vinblastine Zorbax XDB C18 Methanol and Nano pure water with MS Isocratic 4.91–5.2 1 Leaves 90% Ethanolic Ayob et al. (2017)

Journal of Ethnopharmacology 284 (2022) 114647

(150 × 4 mm, 5 0.01% acetic acid
mμ)
Ajmalicine, VP-ODS C18 (200 (A) 0.04% HoAc− H2O, (B) 0.04% MS – 0.5 Leaves 70% methanolic Zhang et al. (2017)
serpentine, × 150 mm, 5.0 HoAc− MeCN
vinblastine and μm)
vincristine
22 alkaloids C18 (2.1 × 50 mm, Water (solvent A) and acetonitrile MS Gradient 1.8–13.4 0.4 Root, stem, 70% Methanolic Jeong and Lim
1.7 μm) (solvent B) containing 0.1% formic and leaf (2018)
acid ammonium formate
61 alkaloids Thermo Вsil C8 using 0.1% formic acid in water and MS Gradient 1–37 min 0.5 Leaf, stem Ethanolic Kumar et al.
(250 × 4.5 mm, 5 acetonitrile and root) (2018a)
μm)
0.1% aqueous FA and acet-onitrile HPLC-MS Gradient 0.6 leaves Choline chloride, Koel et al. (2020)
(containing 0.1% FA) Menthol
(continued on next page)
 S. Kumar et al.
Table 4 (continued )
S.N. Instrument Analytes Column Mobile Phase Detection Elution Range of RT Flow Plant part Extract Reference
program (min) rate
(mL/
min)

vincamine, Eclipse Plus C18

catharanthine and (150 × 2.1 mm,
vindoline 3.5 μm)
18 phenolics LiChroCART C18 1% Acetic acid (A) and acetonitrile UV, MS Gradient 1.0–30.0 1 Stems, leaves Aqueous Ferreres and Pereira
(250 × 4 mm, 5 (B) and petals (2008)
μm) (Merck,
Darmstadt,
Germany)
7,3-O- Kinetex C18 (A) 0.1% formic acid (v/v) in water; LC–MS Gradient 0–20.5 0.3 Flower petals Natural deep Dai et al. (2016)
dimethylcyanidin, (B) 0.1% (v/v) formic acid in eutectic solvents
hirsutidin 3-O-(6-O- methanol
p-coumaroyl)
glucose, 7-O-
methylcyanidin,
malvidin 3-O-(6-O-p-
coumaroyl) glucose
and petunidin 3-O-(6-
O-p-coumaroyl)
glucose
29

4. UPLC-MS Vindogentianine Waters XBridge A (0.1% formic acid in water) and B MS Isocratic – 1.5 Leaves Dichloromethane Tiong et al. (2015)
C18 (50 × 2.1 mm, (0.1% formic acid in MeOH) and methanol
2.5 μm)
5. UHPLC– Ajmaline, ajmalicine, ACQUITY UPLC acetonitrile, methanol and formic MS Gradient 2.09–5.92 0.5 Leaf, stem Ethanolic Kumar et al.
MS serpentine, reserpine, BEHTM C18 (2.1 acid root) (2018b)
vinblastine, × 50 mm, 1.7 μm)
vincristine,
vindesine, vindoline,
vinpocetine and
yohimbine
6. CE-MS Catharanthine, (6500 × 50 μm) 20 mM ammonium acetate and MS Directly 13–17 0.4 Stem Methanolic Chen et al. (2011)
vinblastine and (Yongnian, Hebei, 1.5% acetic acid injected
vindoline China)

Journal of Ethnopharmacology 284 (2022) 114647

S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Table 5
Pharmacological activities and key results of different extract of plant parts of C. roseus.
S. Pharmacological Compounds/plant Extract/Class of compound Dose Result Reference
N. activity part

Anticancer and Leaves Ajmalicine, vindorosine, – Ajmalicine and vindorosine reports for the Tin-Wa et al. (1968)
cytotoxic lochnerinine cytotoxic alkaloid
1. Leaves Aqueous 2.55, 2.38 μg/ The crude aqueous extract of C. roseus shows Ahmed et al. (2010b)
mL differential effects of inhibiting the proliferation
in different cell lines
Leaves Catharanthine 60 μg/mL Catharanthine showed the most considerable Siddiqui et al. (2010)
activity
Stem Methanol, n-butanol, aqueous 5.2–21.0 μg/mL n-butanol fraction prepared from C. roseus stem Pham et al. (2018)
fraction antitumor therapeutic agents.
suspension Indole alkaloid-enriched 211, 210 ng/mL Indole alkaloid-enriched bioactive extract is Fernández-Pérez
cultured-cells bioactive extract powerful antitumor activity et al. (2013)
Whole plants Dimeric indole alkaloids – All these compounds were evaluated for their in Wang et al. (2012b)
vitro cytotoxic activities against human breast
cancer cell line.
– 50 alkaloids 0.1–1 μg/mL All alkaloid fractions showed a dose-dependent Khanavi et al. (2010)
cytotoxic effect on the different cell lines.
– Vinblastine, vincristine – Vinblastine, vincristine showed ctotoxic effects. Ruszkowska et al.
(2003)
2. Antidiabetic Leaves and twigs Dichloromethane:methanol 500 mg/kg Results showed increased metabolization of Singh et al. (2001)
extract (1:1) glucose in treated rats.
Leaves Leaf juice or water decoction 0.5, 0.75, 1.0 The leaf juice showed significant antidiabetic Nammi et al. (2003)
mL/kg BW activity.
Leaves Ethanol 150, 300 mg/kg C. roseus showed lowering blood glucose level. Akhtar et al. (2007)
Leaves Aqueous extract 100 & 500 mg/ The aqueous extract showed significant Mostofa et al. (2007)
kg antidiabetic activity
Leaves Ethanol 30 mg/155 ± 15 C. roseus leaves extract treated animals have Ara et al. (2009)
shown the hypolipidemic effects.
Leaves Petroleum ether, chloroform, 150 mg/kg Chloroform fractions of C. roseus showed high Islam et al. (2009b)
ethyl acetate fractions antiyperglycemic activity
Leaves Aqueous 3, 500 mg/BW 500 mg dose showed significant effects. Prasad et al. (2009)
Leaves Petroleum-ether, ethyl 150 mg/kg Ethyl acetate fractions were the best in activity. Islam et al. (2009a)
acetate, chloroform fractions
Leaves Methanol, n-hexane, 200 μg/mL Chloroform fraction showed the highest activity Siddiqui et al. (2010)
chloroform, water
Leaves Dichloromethane: methanol 500 mg/BW Leaf dichloromethane methanol extract was Jayanthi et al. (2010)
found to exhibit a significant anti hyperglycemic
activity.
Leaves Powder 55 mg/kg BW Potential hypolipidemic properties reported in Rasineni et al. (2010)
leaf extract.
Leaves Powder 100 mg/kg BW C. roseus with its antidiabetic properties could Rasineni et al. (2010)
be a potential herbal medicine in treating
diabetes.
Leaves Aqueous extract 0.30, 1 mL/kg The study showed significant reduction glucose. Ahmed et al. (2010b)
BW
Leaves – 100 mg/kg BW C. roseus showed potent hypolipidemic Rasineni et al. (2010)
properties
Leaves Ethanolic 200, 500 mg/kg Ethanol extract of C. roseus possesses significant Hassan et al. (2011)
antidiarrheal effect.
Leaves – 150 mg/kg BW C. roseus leaves have potent antihyperglycemic Manoharan et al.
effect in alloxan-induced diabetic rats. (2011)
Leaves Methanol 250 mg/kg The leaf extract of C. roseus significantly Ohadoma and
increases the hypoglycemic effect. Michael (2011)
Leaves Leaf juice 0.5, 1 mL/kg Leaf juice of C. roseus possesses significant lipid Patel et al. (2011)
lowering and anti-atherosclerotic activity
Leaves Aqueous 250 mg/Kg The best hypoglycemic activity was found in Vega-Ávila et al.
aqueous extracts. (2012)
Leaves Total phenolic contents – The highest inhibitory effect of 63% is shown by Aadil et al. (2012)
phenolic extract.
Leaves Ethanol 20 mg/kg Ethanolic extract C. roseus exhibited Ahmed and Rao
hepatoprotective activity (2013)
Leaves Vindoline, vindolidine, 25.0 μg/mL Vindolicine induced relatively high glucose Tiong et al. (2013)
vindolicine and vindolinine uptake.
Leaves Aqueous 10 mg/100 g Aqueous extract was found to be effective as an Muralidharan (2014)
antidiabetic agent.
Leaves – – C. roseus fresh leaves showed hypoglycemic Bisla et al. (2014)
activity.
Leaves Aqueous 150 mg/kg Leaves were inactive for hypoglycaemia effect Muralidharan (2014)
Leaves Methanolic 200, 400 mg/kg The methanolic extract of C. roseus significantly Singh et al. (2014)
BW increases the wound contraction in mice that
promises to overcome the delayed wound
healing in diabetic condition.
Leaves Ethanolic 100, 200, Antidiabetic effect of C. roseus on STZ induced Al-Shaqha et al.
diabetes. (2015)
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30
S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Table 5 (continued )
S. Pharmacological Compounds/plant Extract/Class of compound Dose Result Reference
N. activity part

Leaves Ethanol 150 mg/kg Ethanolic extract of C. roseus leaves exhibit Mohan et al. (2015)
antidiabetic activity
Leaves Vindogentianine 200 μg/mL In vitro α-amylase and α-glucosidase inhibition Tiong et al. (2015)
assay showed low inhibition under treatment of
vindogentianine
Leaves Aqueous – Leaf extracts of C. roseus has hipolipidemic Suarsana et al.
activity in hypercholesterolemic rats. (2015)
Leaves Methanol 250 mg/kg BW Methanolic extract of leaf of C. roseus reduce the Aruljothi and
blood glucose level to the rats. Samipillai (2016)
Leaf and flower Ethanol 12.5 mg/mL The alcoholic extract has a main role in Malathi et al. (2010)
carbohydrate metabolism like α-amylase and
α-glucosidase.
Leaves and twigs Total alkaloids extract 100–150 mg/kg The results showed alkaloids extract possessed Zhang et al. (2016)
in antiyperglycemic activity againt STZ induced
hyperglycemic rats.
Leaves, flowers Aqueous 25 mg/mL Aqueous extracts of C. roseus showed Iweala and Okeke
and tender stems considerable hypoglycemic effect. (2005)
Whole plant Methanolic 300, 500 mg/kg The methanolic extract at high dose (500 mg/ Ahmed et al. (2010b)
kg) showed significant antidiabetic activity
3. Antimicrobial Crude extracts/ Ethanolic, methanolic, 100 μg/mL Ethanol showed highest activity whereas Ramya (2008)
leaves, stem, root aqueous aqueous extract was inactive
and flower
Callus Aqueous extract of callus – C. roseus callus extract showed significant Batra et al. (2004)
antibacterial effect.
Hairy roots/wild- Methanolic 100–300 μg/mL High antimicrobial activity reported in the Hanafy et al. (2016)
type roots extracts of the transgenic hairy roots
Leaf, nodal callus, Methanolic – Methanolic extracts possess a great strength to Naz et al. (2015)
fruit fight against the microbial activity
Leaves Ethanol – Leaf extract possesses antibacterial activity Khalil (2012)
Leaves Acetone, ethanol,,chloroform 100 mg/mL Acetone, ethanol and chloroform extracts have Shanmugaraju and
inhibitory effect against most of the pathogenic Bhakyaraj (2016)
microorganisms.
Leaves Methanol ethanol acetone – Leaf extracts show significant antimicrobial Patil and Ghosh
activity (2010)
Leaves Ethanol, acetone, aqueous 25–50 μg/μL All extracts showed significant inhibition Kumari and Gupta
against selected microbes. (2013)
Leaves Aqueous, ethanolic/its 5, 10, 20, 40, 80 Highest antifungal activity was found in Roy & Chatterjee
fractions of petroleum ether, mg/mL chloroform fraction (2010)
diethyl ether, chloroform
Leaves Water, ethanol, methanol 25–100 μg/disc Leaf extracts having the potential to inhibit the Balaabirami &
acetone, hexane growth of bacteria and fungi. Patharajan (2012)
Leaves, shoot, and Alkaloids – Antimicrobial activity against Vardhan et al. (2012)
root multiphytopathoges
Leaves, stem, Ethanol and methanol 100–300 μg/mL The ethanolic extract showed potential Govindasamy and
flower and root antibacterial activity. Srinivasan (2012)
Leaves, stem, 95% ethanol The root extract was most effective which Raza et al. (2009)
flower, root exhibited broad-spectrum antibacterial activity
Leaves, stems and Ethanolic, methanolic, – Ethanolic extract showed high antibacterial Chaman et al. (2013)
flowers aqueous, chloroform while methanolic extracts reported for high
antifungal activity.
Leaves, stems, Methanol, acetone, ethyl 250–1000 mg/L Ethyl acetate extracts of different plant parts Sathiya et al. (2008)
roots and flowers acetate were found to possess best antibiogram followed
by methanol and acetone extracts.
Root Hexane, chloroform, ethyl 250–1000 μg/ Chloroform extract was found to be most active Ethalsha and Retna
acetate, methanol, aqueous mL amongst tested extracts (2014)
Seeds – Antibacterial activities were observed in the Kumar et al. (1997)
seeds of C. roseus
Stem Methanol/its fractions n- – n-Butanol fraction showed highest antimicrobial Pham et al. (2018)
butanol, n-hexane, aqueous activity
Water, methanol – Methanol extract was found to be the best for Kabesh et al. (2015)
antibacterial activity.
4. Antioxidant Cell suspensions Crude alkaloid 50 μg/mL This crude alkaloid extract showed strong Verma et al. (2013)
activity
Leaf callus Compound RF1 1.0 mg/mL Compound RF1 was found to possess strong Verma et al. (2012)
cultures antioxidant potential.
Leaf, stem, flower, Water, methanol, ethanol, – Water extract of leaf shows high antioxidant Pham et al. (2018)
and root ethyl acetate activity.
Leaves Methanol, aqueous 50–250 μg/mL It exhibited potent free radical scavenging and Aadil et al. (2012)
antioxidant activity.
Leaves Vindoline, vindolidine, 0–25.0 μg/mL Vindolicine highest antioxidant potential. Tiong et al. (2013)
Vindolicine, vindolinine
Leaves Ethanol 55.2, 26.22, Ethanol extract of C. roseus does not have Widowati et al.
728.05, 404.76 antioxidant activity. (2011)
μg/mL
Leaves and twigs Total alkaloids 150 mg/kg CTA showed strong antioxidant effect Zhang et al. (2015)
(continued on next page)

31
S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Table 5 (continued )
S. Pharmacological Compounds/plant Extract/Class of compound Dose Result Reference
N. activity part

Root Hexane, chloroform, ethyl 100–1000 μg/ Crude chloroform extract of C. roseus showed Ethalsha & Retna
acetate, methanol, aqueous. mL high vitro antioxidant activity (2014)
Root and shoot – 0, 50, 100 μM. This plant shows good antioxidant potential. Rai et al. (2014)
Root, leaf, hairy Ethanol 200–400 μg/mL The hairy roots of C. roseus showed considerable Mardani-Nejad et al.
root and callus antioxidant power. (2016)
Roots Ethyl acetate extracts 500–1000 μg/ It shows significant antioxidant activity and Jasmine & Agastian
mL α-glucosidase inhibition (2013)
Roots Aqueous – C. roseus was very good source of natural Pereira et al. (2010)
antioxidants
Seeds, stems, Aqueous extracts 300–1200 μg/ A concentration-dependent protective effect was Ferreres and Pereira
leaves and petals mL observed for seeds and tissues, with petals (2008)
shown to be the most active.
Shoots N–hexane, ethylacetate, 20–100% 100% methanolic extract and 100% ethyl Rasool et al. (2011)
methanol, chloroform acetate fraction of C. roseus shoots exhibit
highest antioxidant activity.
Stem Methanol, n-butanol (b); and – n-Butanol fraction possessed the strongest Pham et al. (2018)
aqueous fraction antioxidant capacity amongst the tested
extracts.
Whole plant Ethanol 125; 62.5; 31.25; C. roseus extract had no antioxidant property. Widowati et al.
15.625; 7.813, (2010)
3.906 μg/mL
5. Larvicidal & Leaves Aqueous, ethyl acetate and 50 mg/mL The aqueous and methanol extracts of C. roseus Subarani et al.
pupicidal methanol leaves had an excellent potential to control the (2013)
malarial vector
Leaves Petroleum ether 20 g/L crude extracts of C. roseus reported to possessed Panneerselvam et al.
the larvicidal potentiality. (2013)
Leaves Aqueous, methanol crude, 1000 ppm All extracts exhibited moderate larvicidal Alaguchamy and
petroleum ether, methanol effects. Jayakumararaj
fraction, ethyl acetate fraction (2015)
Leaves Hexane, ethyl acetate, and – The organic leaf extract of C. roseus showed Pavunraj et al.
methanol significant larvicidal effects. (2017)
Leaves n-hexane fraction of acetone 1 ppm The highest concentration produced 100% Kuppusamy et al.
extract mortality in first to second instars. (2009)
Root Silver nanoparticles 3.5–5.0 mg/mL C. roseus offer potential source for larvicidal Rajagopal et al.
activity against the larvae of both dengue and (2015)
filariasis vectors.

S.N.: Serial Number; BW: body weight; ppm: parts per million.

standard deviation %) and 10.9–10.5 (RE: relative error %). The re­ simultaneous quantitation of MIAs in ethanolic extracts of 39 samples of
covery rate of the alkaloids of samples varies from 79.9 to 91.5%. C. roseus collected from five different locations in India (Kumar et al.,
Inter-day precision and stability of samples showed that the compounds 2018b). Compound 95 was found one of the most abundant compounds
111, 112 and 131 were stable at room temperature, 4 ◦ C and − 20 ◦ C for in C. roseus may be due to conversion of compound 80 to 95 is very rapid
2 h, 12 h and two weeks, respectively. The developed method was as previously reported methods (Kumar et al., 2018b; Jeong and Lim,
applied for quantitation of MIAs and validated with three plant parts of 2018; Liu et al., 2007).
three batches of C. roseus collected from three regions of China (Uniyal
et al., 2001). Similar methodology, the ultra-performance liquid
5.2. Qualitative and quantitative analysis of flavonoids and phenolic
chromatography-quadrupole time-of-flight (UPLC-Q-TOF) mass spec­
acids
trometry method was reported Jeong and Lim (2018) who claimed that
method more sensitive, rapid and novel for quantitation of compounds
Piovan and FilippiniFavretto (1998) and Mustafa and Verpoorte
27, 34, 80, 95, 111 and 112 in extracts of C. roseus. The C18 column has
(2007) have reviewed analytical methods reported for the analysis of
been used for the separation of these alkaloids by mobile phase aceto­
phenolics. Among all ionization sources available, ESI in negative ion
nitrile and water with 0.1% formic acid and 10 mM ammonium acetate
mode is regarded as the most suitable for the analysis of phenolics. The
within 13 min. Compounds 111 and 112 were detected with higher
analysis of phenolics (flavonoids and phenolic acids) was reported by
abundance in the aerial parts whereas the roots showed high content of
both UV–Vis and MS with detection limits for flavonoids in the range of
compounds 27, 34, 80 and 95 (Jeong and Lim, 2018). Similarly, the
10 ng whereas single ion monitoring (SIM) mode provides better
HPLC-ESI-MS/MS method has been reported for the quantitation of
detection limits, usually below 1 ng. One drawback in SIM mode,
selected alkaloids present in trace amounts in C. roseus. The content of
however, causes loss of valuable information concerning fragmentation
compound 131 was reported 2.4–17.6 μg/g, DW (dry weight) in leaf,
patterns, which is very important for the identification and character­
stem, and root of C. roseus samples collected from three different regions
ization of various compounds from plant extracts (Ren et al., 2018;
of China (Zhang et al., 2014). In our findings, sensitive, rapid, validated
Gumbi et al., 2019). Therefore, the phenolic compounds of C. roseus
and reproducible ultra-high-performance liquid chromatography-
have been analyzed using HPLC, HPLC-DAD-ESI-MS, and
electrospray ionization tandem mass spectrometry (UHPLC-E­
HPLC-DAD-ESI-MS/MS (MustafaVerpoorte, 2007; Parthasarathy et al.,
SI-MS/MS) method has been published for simultaneous quantitation of
2020). Piovan and FilippiniFavretto (1998) were studied to understand
compounds 27, 52, 72, yohimbine (77), 78, 80, 95, 111, 112 and 121 in
the distribution of these metabolites within the different plant tissues of
crude extracts of C. roseus in mode using ultra-high-performance liquid
leaves, stems, seeds, and flowers. Fifteen glycosides of isorhamnetin,
chromatography coupled with electrospray ionization hybrid triple
quercetin and kaempferol have been identified and characterized, some
quadrupole-linear ion trap mass spectrometry (UHPLC-E­
of them quantified in extracts of these plants using HPLC and HPLC-MS.
SI-QqQLIT-MS/MS) in MRM mode. The method was applied for the
The synthesis of bioactive compounds depended upon the agricultural

32
S. Kumar et al.
practices, climate, or life cycle of the plants, therefore compound 162
was not detected in some studies reported by Ferreres and Pereira
(2008). Crude extract of roots of C. roseus have been also examined for
the presence of phenolic compounds, but no such phenolics have been
detected (Ferreres and Pereira, 2008). The two main flavonoids 154 and
150 have been reported in C. roseus leaves and stems (Nishibe et al.,
1996; MustafaVerpoorte, 2007). The compound 154 was reported to be
abundant in petals, while compound 149 also detected in the petals.
Another isomer with a similar fragmentation was compound 156
detected only in trace amounts in the stems and leaves (Cui et al., 2000).
Compound 151 has been reported solely in petals in trace amounts.
Compound 155 has been reported in higher amounts in stems, seeds,
and petals, whereas compound 156 found in seeds of C. roseus. Com­
pounds 155 and 156 were detected in very low amounts in seeds,
whereas petals showed an abundant amount of compounds 149, 156,
155, 159 and 160 (Steinmann and Ganzera, 2011; Simirgiotis et al.,
2012). Compound 154 was the most abundant in seeds. Compound 154
was detected 292.3 mg/kg, DW, and 52.7 mg/kg DW in seeds and leaves,
respectively. The amount of compound 155 was reported in 2.0, 714.2,
190.8, 8.5, 8, 120.8 mg/kg, DW in seeds, stems, leaves and petals
respectively. Similarly, compound 149 was detected in the amounts
582.7, 190.5, 310.9, 1027.9 mg/kg, DW in seeds, stems, leaves, and
petals respectively (MustafaVerpoorte, 2007).
High concentrations of anthocyanins (0.001–0.50 μmol/g fresh
weight) were reported in all cells present in a medium induced culture.
The compounds 170–172 have been identified and characterized as
aglycones of the anthocyanins. The total phenolic content was detected
ranged from 20.0 to 70.0725 mg/g fresh weight) (Knobloch et al., 1982).
The anthocyanin content in the flowers from both types of plant were
comparable and approximately 1.5 times higher than those obtained
from cell suspensions. Compound 178 was the major compound
(49.3–70 relatively abundant) in this plant. The extracts from both
embryogenic plant flowers and cell cultures have contained the malvidin
derivatives in a higher amount than the petunidin ones (Filippini et al.,
2003). Compounds 174, 175, 176, 177, 178 and 179 have been
detected at 3.50, 1.50, 11.50, 28.0, 49.3 and 6.20 mg/g, fresh weight
respectively using direct infusion electrospray (ESI) mass spectrometry
and ion trap multiple mass spectrometry in positive-ionization mode.
The all three 3-O-(6-O-p-coumaroyl) glucosides were identified and
characterized for the first time in C. roseus (Piovan and FilippiniFavretto,
1998).
5.3. Qualitative and quantitative analysis of volatile compounds
The volatile compounds have identified by chromatography-mass
spectrometry (GC-MS) in leaves, stems, flowers, and roots of C. roseus.
Mohan et al. (2015) has identified eleven compounds in the ethanolic
extract of the leaves and whereas fifteen compounds have been reported
by Doshi et al. (2018) in the same extracts. Seventy six compounds have
been identified in aqueous extract of the leaves and industrial marcs
(Brun et al., 2001). The high of the content volatile compounds has been
reported in essential oils of the leaves consisting mainly of linolenic acid
ethyl ester (43.9%) (330), phytol (29.6%) (274), limonene (23.2%)
(212), stearic acid (10.6%) (314), geraniol (7.3%) (278) and dodecyl
alcohol (9.8%) (271) (Lawal et al., 2015). Pandey-Rai et al. (2006) re­
ported fifty two compounds and accounting showed 91.3% volatile of
the leaf oil. Eighteen compounds have been identified and assessed the
abundances of these compounds in each cultivar. The result showed
abundance of each cultivar as dark pink (66%), pink (50%), pinkish red
(55%), milky white (50%), whitish pink (50%), white (44%), purple
pink (44%) and pale pink (27%) (Fitrianto et al., 2020).
6. Pharmacological activities
Various extracts and isolated compounds of C. roseus has been re­
ported a wide range of pharmacological activities such as anticancer and
33
Journal of Ethnopharmacology 284 (2022) 114647
cytotoxic, antidiabetic, antimicrobial, antimicrobial, antioxidant larvi­
cidal and pupicidal (Gajalakshmi et al., 2013; Nisar et al., 2016; Das and
Sharangi, 2017; Mishra and Verma, 2017; Senbagalakshmi et al., 2017;
Paarakh et al., 2019). Among them the anticancer activity is well known
for this plant. The major pharmacological activities and their method­
ology such as type of extraction, plant parts used, dose and key results
which were used during assessment are summarized in Table 5.
6.1. Anticancer activity (cytotoxic activity)
The different extracts of leaves of C. roseus showed significant anti­
cancer activity due to the presence of its bisindole alkaloids (Mishra and
Verma, 2017; Senbagalakshmi et al., 2017; Paarakh et al., 2019). The
characteristics and major active compounds 112 and 111 of the plants
used in chemotherapy for treatment of many types of cancer (Orwa
et al., 2007). In contrast, these compounds isolated from the various
extracts of the plants have been reported for cytotoxicity in a wide range
of cell lines. The methanolic extract of C. roseus has been reported for
cytotoxicity against human colorectal carcinoma cell line (HCT 116)
using MTT assay. The highest activity dose-independent cytotoxicity
reported in chloroform and methanol fractions. The n-hexane fraction
showed moderate whereas minor cytotoxic activity occurs in water
fraction (Sagar et al., 2020). Compound 27 has been reported for cyto­
toxic activity at dose at 200 μg/mL, whereas the most promising cyto­
toxic activity has been observed in compound 55 at the dose-dependent
manner with IC50 value of 60 μg mL (Siddiqui et al., 2010). Three
fractions of the alkaloidal extract have been reported for cytotoxic ef­
fects on cell proliferation of HT-29, Caco-2, T47D and NIH/3T3 cell lines
due to dose-dependent cytotoxicity. The proliferation of NIH/3T3 cells
has been less affected in comparison to other cell lines (Khanavi et al.,
2010). The n-butanol fraction have been reported for in vitro cytotoxic
activities on cell line Du145 (prostate), HT29 (colon), H460 (lung),
A431 (skin), A2780 (ovarian), MIA PaCa-2 (pancreas), MCF-7 (breast
SJ-G2, U87), BE2-C (neuroblastoma) and SMA (glioblastoma) at low
doses (GI50 values of 5.2–21.0 μg/mL) (Pham et al., 2018). The indole
alkaloid-enriched bioactive extract with methyl jasmonate (MJ) and
cyclodextrins (CDs) has been produced cytotoxicity in three cell lines,
such as THP-1 human monocytic leukemia, Jurkat E.6 human lympho­
cytic leukemia and BL 1395 non-tumor human B-cell line. The cytotoxic
assays have been studied using the colorimetric assay 2, 3-bis
(2-methoxy-4-nitro-5-sulfophenyl)-S-[(phenylamino) carbonyl]-2 tetra­
zolium hydroxide (XTT). The Jurkat E.6 and THP-1 cell lines inhibited
by 50% were 211 and 210 ng/mL, respectively. Fernández-Pérez et al.
(2013) claimed that the extract has been exhibited the potent antitumor
activity due to the effect of compounds 111 and 112 but depends on the
synergistic effect of the compounds 6, 55, 80 and lochnericine (16). The
C. roseus extract has significantly induced the proliferation of PBMCs (P
< 0.01) taken from normal donors comparing Jurkat cells for cytotoxic
effects. The maximum cell proliferation was reported in PBMCs incu­
bated with 1000 μg/mL of the C. roseus extract with increased viability
of 165.93% at 24 h, 117.67% at 48 h and 116.67% at 72 h. The distinct
effects of inhibition of proliferation of the Jurkat cell line and stimu­
lating the growth of PBMCs due to aqueous extract of C. roseus (Ahmad
et al., 2010a). The cytotoxic activities of eight bisindole alkaloids
including, catharine (122), leurosidine ( 123), compound 125 and
vinamidine (135) etc have been reported on MDA-MB-231 cells were
tested by MTT assay. These alkaloids might induce a marked reduction
of the cell viability after 72 h treatment. Among these, the compound
125 has exhibited the most potent inhibitory activity, while compound
135 has been found the weakest activity. It is found that compound 123
showed much weaker activity than that of compound 111. The
17-deacetoxy derivatives of compounds 111, 112, 135, 221 and 222
have a more potent activity than compound 135, implying the lack of
acetoxy at C-17 seems to be an advantage in increasing cytotoxicity.
These results provided clear indication for further structural modifica­
tion in bisindole alkaloids (Rosenthal and Kaufman, 1974; Gomber et al.,
S. Kumar et al.
2010; Wang et al., 2012b; Parthasarathy et al., 2020).
6.2. Antidiabetic activity
Diabetes is metabolic disorder which are characterized as abnormal
enhancement of the glucose level in endocrine system. Few studies
carried out by Muralidharan (2014) and Aruljothi and Samipillai (2016)
reported that the application of aqueous extract of C. roseus leaf in
alloxan-induced diabetic rats has significantly (P < 0.001) decreases the
blood glucose levels. Finding also reported that total glycerides (TC),
low-density lipoprotein (LDL), very low density lipoprotein (VLDL) and
triglyceride (TG) decresease and comes to normal by using C. roseus leaf
extract. No deaths have been reported in 30 days of the experimental
period and therefore, it is a good plant species for treatment of diabetes.
In another study, the aqueous herbal extract preparation containing
C. roseus at a dose rate of 1 g/kg, A. indica (500 mg/kg), and A. sativum
(1 g/kg) BW has been administered in rats orally for 14 days, and finding
suggested that the extract showed significant reduction of blood glucose
as compared to patent drug (Mostofa et al., 2007). Similarly, the
comparative study of some plant species of other genus have been re­
ported using the aqueous leaf extract of M. koenigii, P. guajava and
C. roseus at dose of 500 mg/kg treated in streptozotocin-induced diabetic
rats (SIDRs) was carried out by different co-workers. They are reported
significant body weight gain and blood glucose level in the
streptozotocin-induced diabetic rats (SIDRs) after fifteen days of treat­
ment (M. koenigii and P. guajava: (P < 0.05), C. roseus, and GBC: (P <
0.001). Findings suggested the aqueous leaves extract of M. koenigii,
P. guajava and C. roseus possessed beneficial effects in diabetic mani­
festations (Prasad et al., 2009; Iweala and Okeke, 2005; Zhang et al.,
2016). Allied to these studies, Islam et al. (2009a) reported for fractions
of petroleum-ether and ethyl acetate significantly reduced blood glucose
levels in normoglycemic rats (RGRs) and SIDRs. The antidiabetic and
hypolipidemic activities have been compared to metformin HCl at the
dose of 150 mg/kg. The C. roseus fractions in ethyl acetate found to be
the best for antidiabetic functions (Islam et al., 2009a, 2009b).
Nammi et al. (2003) have been studied the leaf juice of C. roseus and
treated it to normal and diabetic rabbits, and also findings were
compared with the standard drug and glibenclamide. They reported the
reduction of glucose level after treatment by juice in a dose-dependent
reduction in blood glucose of the both normal and diabetic rabbits.
The alcoholic flower and leaf extract was tested for alpha-amylase and
alpha-glucosidase inhibitory activity and found that the leaf extracts has
the maximum inhibitory action at a dose of 10 mg/mL (IC50). These
concluded that the leaves and flowers of C. roseus have been exhibited
the anti-diabetic effect by lowering α-amylase and α-glucosidase
(Malathi et al., 2010).
The methanolic fractions of leaf at a concentration of 200 and 400
mg/kg BW (body weight) has been optimal and induced through
intraperitoneal in mice (Ahmed et al., 2010). The methanolic extract
was significantly increases the contraction of wounds and the formation
of collagen fibers in the mice compared with controls (P < 0.001). A
methanolic fraction of fresh leaves has been treated to rats induced with
alloxan to decrease the glucose level of blood and found that the serum
protein recovered the normal glucose level in rats (Singh et al., 2014).
Al-Shaqha et al. (2015) have been reported the antidiabetic efficacy of
the ethanolic extract and expressions of GLUT-2 and GLUT-4 gene in
SIDRs. The doses administered orally at 100 and 200 mg/kg, destroys
the glucose carrying system of liver within twenty-eight days. A com­
bination of dichloromethane (DCM) with methanol extract of leaves and
twigs in ratio of 1:1 at a concentration of 500 mg/kg given orally for
seven and fifteen days which have been shown 48.6 and 57.6% hypo­
glycemic activity using SIDRs model (Singh et al., 2001; Jayanthi et al.,
2010). The methanolic and aqueous leaf extracts of Acacia arabica and
Murraya koeingii, C. roseus and Rauvolfia serpentina have been studied for
α-amylase inhibition. The maximum α-amylase inhibition and glucose
diffusion rate were found in methanolic extracts of Rauvolfia serpentina
34
Journal of Ethnopharmacology 284 (2022) 114647
(Aadil et al., 2012). Findings also reported that the hepatic and muscle
glycogen content were decreased with C. roseus administration (Rasi­
neni et al., 2010; Mohan et al., 2015).
Compounds 27 and 38 and vindolicine (140) have been reported
better protein tyrosine phosphatase-1B (PTP-1B) inhibition activity
(Tiong et al., 2013); and these also suggest beneficial against
diabetes-type 2 (Bisla et al., 2014). The hypoglycemic activity in
methanol leaf extracts using alloxan model for diabetes in rats. The leaf
extracts have been significantly increasing the hypoglycemic effect of
metformin (Ohadoma and Michael, 2011; Ahmed et al., 2010b).
Vega-Ávila et al. (2012) reported the blood-glucose-lowering properties
of organics (hexane, dichloromethane, and methanol) and aqueous
extract in healthy and alloxan-induced (75 mg/kg) diabetic mice, after
intraperitoneal administration (250 mg/kg BW. The leaf juice of
C. roseus has been studied for the hypolipidemic activity in adult guinea
pigs according to pharmacokinetic parameters. The leaf juice at dose of
0.5 mL/kg reduced the serum lipid parameters. The high-fat diet de­
creases the serum lipid parameters in the tissue-induced leaf juice in the
dose of 0.5 mL/kg (Patel et al., 2011).
6.3. Antifertility effect
Bioefficacy of ethanol leaf extract of three plant species of C. roseus,
Ocimum sanctum, and Lantana camara were tested for fecundity and
fertility of the red cotton bug, Dysdercus koenigii. The oviposition
behavior, fecundity and fertility of the females who emerged from the
treated nymphs were assessed. D. koenigii exhibited a reduction in their
reproductive success in response to the plant extracts. The ethanol
extract of C. roseus was most effective in the suppression of oviposition
among the examined above plants extracts. The fertile mated females lay
eggs, indicating the insemination of the mated females. However, the
percentage of hatchability of the eggs laid by the females was less. The
eggs laid by the females treated with ethanol extract of Osmium leaf was
least fertile. It was suggested that some of the compounds present in the
extracts individually or synergistically alter the fecundity and fertility of
D. koenigii (Kayesth et al., 2017). Similarly, petroleum ether extract of
the leaves of C. roseus inhibited the estrogen-induced gain in the uterine
weight when administered along with estradiol (Gupta, 2009). The
long-term use of the leaves of C. roseus was caused by a reduction in
epididymal sperm count and lowering of serum testosterone. The
aqueous leaf extract was orally administered to the animals daily for 30
days. After 30 days of treatment with extract significantly reduced the
epididymal sperm count and serum testosterone level respectively
(Sukumar and Osmani, 1981; Shaw et al., 2017).
6.4. Antimicrobilal activity
The crude extracts of various plant parts of C. roseus have been re­
ported for antibacterial activity against several bacterial strains by disc
diffusion assay. Among the different plant parts tested, the leaf extract
exhibited significantly higher efficacy (Gajalakshmi et al., 2013; Nisar
et al., 2016; Das and Sharangi, 2017; Mishra and Verma, 2017; Senba­
galakshmi et al., 2017; Paarakh et al., 2019). Similarly, organic solvent
extracts showed higher antibacterial activity in comparison with their
corresponding aqueous extracts (Gajalakshmi et al., 2013; Nisar et al.,
2016). Aqueous extracts of C. roseus generally were inactive against
selected bacterial strains. Ethanol extracts found to have good activity
against the tested bacterial strains. The gram negative (-ve) stains were
found to have more responsive in comparison to gram (+ve) strains of
bacteria (Ramya, 2008; Hanafy et al., 2016; Fitrianto et al., 2020). Be­
sides, the alcoholic extracts (ethanol and methanol) have been tested
against various human pathogens such as Bacillus subtilis, E. coli,
Staphylococcus aureus, Salmonella typhi, and Pseudomonas aeruginosa. The
ethanolic and methanolic of found to have the minimum zone of inhi­
bition (MZI) as 21.15 ± 1.64 mm and 15.61 ± 1.35 mm, respectively,
against the bacterial strain of S. typhi, whereas the MZI observed against
S. Kumar et al.
S. aureus was 06.24 ± 0.69 mm in the fraction of ethanolic extract and
MZI observed in methanolic fraction was 05.20 ± 0.86 mm against
E. coli (Srinivasan et al., 2001; Govindasamy and Srinivasan, 2012).
The agar-well diffusion method was employed to test the antimi­
crobial activity using the ethanolic, methanolic, aqueous and chloro­
form fractions/extracts of different plant parts (flowers, stems, leaves,
roots) of C. roseus against E. coli, P. aeruginosa, S. aureus, Candida albi­
cans, Aspergillus niger and Aspergillus fumigates (Chaman et al., 2013).
The studies concluded the ethanolic extracts of C. roseus found to have
strong properties against C. albicans, P. aeruginosa, and A. niger. Etha­
nolic extracts of stems and flowers exhibited against S. aureus and
A. niger. Chloroform extract of flower has activity against gram-negative
bacteria of P. aeruginosa. The aqueous fractions of leaves, stems and
flowers were not efficient against bacteria and fungus strains (Chaman
et al., 2013). The hexane, chloroform, methanol, ethyl-acetate and
aqueous extracts of root were tested for antibacterial activity against
E. coli, Proteus vulgaris, Klebsiella pneumonia, S. aureus, Micrococcus luteus
and B. subtilis. The chloroform extracts showed high potential against
almost all tested bacteria species. Gram-negative bacterial strains found
to be less sensitive than Gram-positive bacterial strains (Ethalsha and
Retna, 2014). The ethyl acetate extracts of different plant parts showed
best among extracts of acetone and methanol of flowers, leaves, stems,
and roots for antibiogram followed by acetone and methanol extracts.
The extracts of all plant parts were observed the largest antibiogram
against the bacterial strain B. subtilis followed by K. pneumonia species,
whereas the smallest antibiogram was found against the species of
Streptococcus strains. The S. aureus strain was found to be responsive in
different plant extracts of C. roseus. The ethyl-acetate extract contributed
as the best antibiogram to high solubility which was due to the presence
of bioactives of C. roseus than other solvents used for the study (Sathiya
et al., 2008). A study carried out by Kumar et al. (1997) have been re­
ported screening the seeds of 40 plant species including C. roseus for
their antibacterial properties using the strains of B. subtilis, E. coli,
Pseudomonas cichorii and Salmonella typhimurium, and found the seeds of
C. roseus as the most active plants for antibacterial activity. P. cichorii
recorded mortlity of 97%, followed by B. subtilis (83%), S. typhimurium
(69%) and E. coli (55%) bacterial strains. C. roseus extracts antimicrobial
activity have been carried out and tested using pathogenic bacterial
strains (Bacillus licheniformis, B. subtilis, Azotobacter chroococcum) using
the agar well diffusion method. In vivo and in vitro studies of leaf and in
vitro study of callus of leaves, fruits and nodal explants in methanol has
been exhibited good antibacterial activity (Naz et al., 2015). The ethanol
extract of leaf found to have showed very strong antibacterial activity
against a few human pathogenic microorganisms (S. aureus and E.coli) as
well as pathogenic fungi (C. albicans) by the disc diffusion method. The
100 mg/mL leaf extract found to have the strongest inhibition activity
against S. aureus (15 mm MZI) followed by E.coli (11 mm MZI) (Balaa­
birami & Patharajan, 2012).
In another study, the antimicrobial activity have been reported from
the C. roseus leaf extract by Balaabirami and Patharajan (2012) using
microorganisms such as E. coli, Klebsiella oxytoca, K. pneumoniae, Proteus
mirabilis, P. aeruginosa, S. typhimurium, Salmonella paratyphi, Shigella
boydii, S. aureus by disc diffusion method. The result shows that leaf
extracts have the good potential to inhibit the active growth of bacterial
strains. C. roseus extracts did not exhibit antibacterial activity against
S. aureus, whereas, it was also ineffective against P. aeruginosa. On the
other hand, C. roseus stem crude extract was inactive against Shigella
boydii. The C. roseus root extract was the most effective against S. typhi
and S. boydii with the MZI determined 24 mm and 22 mm, respectively.
Raza et al. (2009) have been tested flower extract against Corynebacte­
rium diphtheriae and found to possess a significant antibacterial activity.
A study by Shanmugaraju and Bhakyaraj (2016) found that ethanolic
extract exhibited the maximum antibacterial activity in comparison to
the C. roseus leaf extract prepared in acetone and chloroform. The tested
bacteria S. aureus were susceptible to leaf extracts than tested Escherichia
coli. The antimicrobial activities of ethanol, acetone and methanol
35
Journal of Ethnopharmacology 284 (2022) 114647
extracts have been checked against P. aeruginosa, S. typhimurium, and
S. aureus. All these extracts showed antimicrobial activity against
selected microorganisms (Patil and Ghosh, 2010; Kabesh et al., 2015).
The C. roseus n-butanol extract/fraction was observed effectively
inhibiting the activity of E. coli and Staphylococcus lugdunensis (Pham
et al., 2018).
The ethanolic extracts of leaf showed significant inhibition and
indicated excellent anfungal activity against F. moniliforme in extracts.
MIC values of the extract against the tested fungal strains range between
25 μg/μl to 50 μg/μl (Kumari and Gupta, 2013). Aqueous ethanolic
extract and its different fractions of leaves of C. roseus have been
examined for their antifungal activity. The highest antifungal activity
was found in the chloroform fraction of C. roseus (Roy and Chatterjee,
2010). The high antifungal activity has been reported against C. albicans
by methanolic flower extract of C. roseus followed by methanolic leaf
extract and then methanolic stem extract. In vitro extracts showed better
antifungal activity than in vivo extracts due to MZI (30.3 mm ± 0.58a) by
MIC value is 2.0 mg/mL against B. licheniformis. A similar study of Khalil
(2012) reported the strongest fungal inhibition activity against the
pathogenic fungi C. albicans (12 mm MZI) using the C. roseus ethanlic
leaf extracts. Ethanolic extract showed high antibacterial while meth­
anolic extracts reported for high antifungal activity.
Bacterial intercellular communication, or quorum sensing (QS),
controls the pathogenesis of many medically important organisms. Anti-
QS compounds are known to exist in marine algae and have the ability to
attenuate bacterial pathogenicity. Adonizio et al. (2006) have been
proposed that the terrestrial plants traditionally used as medicines may
also produce anti-QS compounds. 50 medicinal plants from southern
Florida were screened for anti-QS activity using two biomonitor strains,
Chromobacterium violaceum and Agrobacterium tumefaciens. Among them
only six showed QS inhibition: Conocarpus erectus L. (Combretaceae),
Chamaecyce hypericifolia (L.) Millsp. (Euphorbiaceae), Callistemon vim­
inalis (Sol. ex Gaertn.) G. Don (Myrtaceae), Bucida burceras L. (Com­
bretaceae), Tetrazygia bicolor (Mill.) Cogn. (Melastomataceae), and
Quercus virginiana Mill. (Fagaceae). This study introduces not only a new
mode of action and possible validation for traditional plant use, but also
a potentially new therapeutic direction for the treatment of bacterial
infections.
6.5. Antioxidant activity
Antioxidants are protect human body from cancer, inflammatory
disorders and Alzheimer’s diseases (Ethalsha and Retna, 2014). The
different concentrations (100–1000 μg/mL) of chloroform root extracts
of C. roseus reported to have remarkable antioxidant activity using 2,
2-DiPhenyl-1-PicrylHydrazyl radical (DPPH) assay with the IC50
(inhibitory concentration). Chloroform extract has higher antioxidant
potential than standard concentration. The IC50 of C. roseus of extract
has been found at 94.178 μg/mL. Shoots extracts of C. roseus and its
fractions (n-hexane, ethyl acetate, methanol, chloroform) exhibited ac­
tivities related to free radical and percentage inhibition with the linoleic
acid for DPPH. The extracts and their fractions have been determined
inhibition of peroxidation in linoleic acid (38.4–75.1%) along with good
DPPH radical IC50, 28.2–119μg/mL The unconventional antioxidant
activity have been reported in chloroform extracts, it may be presence of
volatile compounds in shoot (Widowati et al., 2010). The generally
aqueous and alcoholic extracts are rich source of flavonoids and
phenolic acids therefore, the shoot extracts of C. roseus and fractions in
100% methanol have been found to exhibt strong antioxidant activity,
and the finding showed significant (p < 0.05) variations (Rasool et al.,
2011). Other reports showed the extracts of C. roseus in methanol have
the highest DPPH scavenging activity than aqueous extracts (Aadil et al.,
2012). Organic acid mixture has been reported from the root extract of
C. roseus and at different concentrations was studied for antioxidant
potential by superoxide, DPPH radicals, and nitric oxide. The effect has
been observed at concentration-dependent up to 19 l g/mL i.e. when
S. Kumar et al.
activity reached its peak. The result indicated that major phytochemical
constituents of plants are not always responsible for the activity, and
organic acids mixture with plant extract also have some protective effect
(Pereira et al., 2010).
A study reported the lyophilised aqueous extract of C. roseus petals
showed the strongest concentration-dependent antioxidant capacity ef­
fects (IC50 at 197 μg-mL), followed by seeds (IC50 at 265 μg-mL) and
leaves (IC50 at 447 μg-mL). The extract prepared from flower petals of
C. roseus has been found to possess concentration-dependent protective
effect (Ferreres and Pereira, 2008). The antioxidant property has been
studied using DPPH and inhibition of malondialdehyde production. The
lowest IC50 for DPPH inhibition found in extract prepared from root
hairs (p ≤ 0.01) than leaf callus and root, leaf, of C. roseus (Mardani-­
Nejad et al., 2016). The water leaf extract had the strongest scavenging
properties of DPPH (22.78 mg/− g), ABTS (35.33 mg/g) and FRAP
(24.18 mg/g), two-fold more of methanol (Pham et al., 2017). The
ethanol and ethyacetate extracts showed lower antioxidant capacity
than H2O over sixfold and ninefold lower, respectively. The drying
procedure has been reported significantly affected the antioxidant
properties of C. roseus. The vacuum drying (VD50) at 50 ◦ C was suitable
for the leaf whereas and the infrared drying (IRD35) at 35 ◦ C was sug­
gested for stem, root, and flower due to phenolic contents (Pham et al.
(2018). The antioxidant properties were studied using the ethanol leaf
extracts of Piper betle, Datura petandra, Curcuma mangga and C. roseus
following DPPH assays. The IC50 values of D. petandra (4.74) and P. betle
(5.49) found to be lowerest than C. roseus (102.96), C. mangga (277.79)
extracts. These findings indicated P. betle and D. petandra extracts are
better antioxidants in comparison to C. roseus and C. manga plant species
(Widowati et al., 2011; Verma et al., 2013). The significant effect was
also reported in C. roseus with the highest ORAC values (2.85 mg of
GAE/g of fresh weight) (Zhao et al., 2001).
6.6. Hepatoprotective activity
Associated liver diseases are considered as the most serious health
problems, as this organ is responsible for metabolism and excretion, and
frequently exposed to various diverse xenobiotics and therapeutic
agents. Ahmed and Rao (2013) have been reported comparative hep­
atoprotective activities of C. rosea, Brassica oleracea, and Melia azedarach
leaves ethanolic extracts with simvastatin induced hepatotoxicity in
rats. Hepatotoxicity induced by simvastatin at a dose of 20 mg/kg for
one month in rats, whereas protected by M. azedarach, C. rosea, and
B. oleracea (300 mg/kg and 500 mg/kg). Hepatotoxicity-induced rats
found to have significant changes in biochemical parameters such as
enhanced serum glutamic pyruvic transaminase (SGPT), serum
glutamic-oxaloacetic transaminase (SGOT), alkaline phosphatase (ALP),
serum bilirubin, and decrease in proteins content. These biochemical
parameters were restored in rats treated with extracts. Finding showed
ethanolic extracts of M. azedarach at a dose of 300 mg/kg and B. oleracea
at a dose of 500 mg/kg found to have potent hepatoprotective properties
in comparison to C. rosea extract. Histopathological studies of liver were
also reported which indicates further confirmation of the hep­
atoprotective effect of the extracts.
6.7. Larvicidal and pupicidal property
C. roseus plants larvicidal as well as pupicidal properties have been
explored by many researchers (Kuppusamy et al., 2009; Sandey and
Summarwar, 2016: Pavunraj et al., 2017). The methanol, aqueous and
ethyl-acetate of leaf extract have been tested against malaria and fila­
riasis vectors such as 4th instar larvae/pupae of Anopheles stephensi and
Culex quinquefasciatus. Results showed that the lethal doses of LC50 and
LC90 at 50% and 90% of the used samples of A. stephensi, C. quinque­
fasciatus which were killed after post treatment of 24 and 48 h. In
aqueous extract, the mortality of larval/pupal was observed after 24 and
48 h, whereas samples treated with ethyl-acetate and methanol extracts
36
Journal of Ethnopharmacology 284 (2022) 114647
showed zero mortality in comparison to control. The larvicidal activity
against the malarial vector A. stephensi was assayed at various concen­
trations of leaf extracts and found to be effective for controlling malaria
vectors (Subarani et al., 2013). In another study, the highest larval
mortality was determined in the petroleum ether extract of C. roseus
against B. thuringiensis due to may be presence of volatile compounds
(Panneerselvam et al., 2013). The larvicidal activity has been evaluated
against larvae with a concentration of 1000 ppm of aqueous extracts.
Finding suggested that the larvicidal activity against gram pod borer
Helicoverpa armigera (Lepidoptera: Noctuidae) increased with larval
development. The moderating larvicidal effects have been observed in
all extracts (2.60, 3.94, 7.75, 30.83, 38.53 and 63.34) to 1st, 2nd, 3rd,
4th, 5th and 6th instar of H. armigera larvae after 24 h of exposure to the
extracts. The aqueous leaf extract has been found ED50 values for their
effect on the larvae of H. armigera and LD50 values were significant at P
< 0.05. Therefore, the aqueous extract of leaves has potential to be use
like biopesticide for H. armigera management (Alaguchamy and Jaya­
kumararaj, 2015). The pupicidal activity of leaf extract was reported
73.33, 49.33, 33.33, 28.00 and 17.33 percent mortality was observed
when pre-pupae were treated with 2.0, 1.5, 1.0, 0.5 and 0.1%, respec­
tively in the pre-pupal stage of Spodoptera litura using topical applica­
tion method. The leaf alcoholic extracts are the good results for
treatment against larvae and pupae (Sandey and Summarwar, 2016).
6.8. Other activities
Compound 52 has been used for Alzheimer’s disease (AD) and de­
mentia populations. This active compound has been reported to be
potent at the doses up to 60 mg/d in clinical trials of dementia and stroke
(Lamar et al., 1988; Krieglstein and Rischke, 1991; Lakics et al., 1995;
Orviský et al., 1997; Gabryel et al., 2002; Manda et al., 2015). The
ethanol extract of the flower of C. roseus has been more strongly stim­
ulating accelerated wound healing activity compared to controls (Nayak
et al., 2007). Tensile strength has been enhanced by hydroxyproline
content and the hypolipidemic active principles due to the influence of
ethanol extract (Senbagalakshmi et al., 2017). The hydroalcoholic or
dichloromethane-methanol extracts of leaves and stem and roots have
shown significant effects in hypotensive (Ara et al., 2009). Hoskeri et al.
(2011) studied in-vitro anthelminthic activity using whole plant parts of
C. roseus in ethanol and water extracts. Different concentrations of
ethanol extract was used for study against P. posthuma. The efficient
paralysis effect (6.67mins) was observed in the ethanol extract at 200
mg/mL than other extracts. Similarly, ethanol extract with dose at 250
mg/mL showed significant anthelmintic activity with a lead time of
46.33 mins (Hoskeri et al., 2011). Sadique and Hashim (2019) investi­
gated the antiangiogenesis activity of C. roseus with ethyl acetate, which
exhibited highly potent antiangiogenesis activity. The efficacy of this
activity may be due to the presence of flavonoids because the ethyl ac­
etate fraction was flavonoid-rich. The antimutagenic activity was
commonly reported in anticancer drugs from medicinal plants and they
contain direct or indirect antimutagens (Banerjee and Mumtaz, 2020).
The antimutagenic actions of triterpenes, hydrocarbons and an alkaloid
isolated from C. roseus has been reported (Inte et al., 1998; Mishra and
Verma, 2017).
The antidiarrheal effect of C. roseus was studied with extracts at 200
and 500 mg/kg doses in castor oil pretreated diarrheal rats. Standard
drug loperamide and atropine sulfate ware used during two experi­
ments. The ethanolic extract has been reported as a dose-dependent
inhibition of castor oil-induced diarrhoea. The most significant differ­
ence was observed at 200 mg/kg (P < 0.05) than at 500 mg/kg (P <
0.001) in the extract with the negative control, which suggested a sig­
nificant antidiarrheal effect (Hassan et al., 2011).
The ethanol extract of leaf has been reported for antiplasmodial ac­
tivity against both chloroquine-sensitive Pf3D7 and chloroquine resis­
tant PfINDO strains of Plasmodium falciparum grown in human red blood
cell cultures with IC50 11–20 μg/mL (Singh et al., 2015). Leaves extract
S. Kumar et al.
can be a good source for synthesis of nanoparticles which shows anti­
plasmodial activity against P. falciparum (Ponarulselvam et al., 2012).
7. Clinical applications
Structures of active leads and drugs that have been identified from
plant parts of C. roseus and currently used for the treatment of various
cancers are presented in 3A, 3B, 4A, 4B. Compound vincaleukoblastine,
VelbeR has been isolated in 1960, used in the treatment of Hodgkin’s
disease, non-Hodgkin lymphomas, testiscarcinomas and sometimes
against breast cancer and chorio-carcinomas (Kris et al., 1985; Levêque
and Jehl, 2007). Compound 112 (leurocristine, OncovinR) is oxidized
form of compound 111 and marketed and commercialized in 1963, and
used against acute leukemia, Hodgkin’s disease, non-Hodgkin lym­
phomas, Wilm’s tumors in children, rhabdomyosarcomas and breast
cancer (Kaplan et al., 1986; Parthasarathy et al., 2020). Anhydrovin­
blastine was recommended for use as an antineoplastic agent in the
treatment of cervical and lung cancer (Quan et al., 2019; Wiernikowski
and Bernhardt, 2020). Some C. roseus alkaloids marketed and
commercialized in 1957 for the treatment of hypertension as pharma­
ceuticals include compound 80 (HydroserpanR, LamuranR) (Senbaga­
lakshmi et al., 2017). This is often considered as the major drug of
R. serpentina (Kumar et al., 2016a, 2016b). The most active bisindole
alkaloids consist of a plumeran (compound 27) and an ibogan (com­
pound 55) moiety (Jacobs et al., 2004; Das and Sharangi, 2017). Besides
alkaloids, other secondary metabolites have been isolated from C. roseus,
including monoterpenoid glucosides (loganin, secologanin, sweroside,
deoxyloganin, dehydrologanin), steroids (catasteron, brassinolides),
phenolics, flavonoids, and anthocyanins showed considerable potential
can be used for further study (Federico et al., 2009; Kabesh et al., 2015;
Zhang et al., 2018).
8. Toxicity
The toxicity of ethanolic, methanolic and aqueous extracts from
plant parts of C. roseus have been inadequately studied (James et al.,
2007). Some studies had toxicity for extracts and isolated compounds at
above the optimized doses. Methanolic extract of leaf studied for liver
and kidney functions in Dawely rats using 0.1, 0.5 and 1.0 g/kg. Dose at
1.0 g/kg causes side effects such as mortality and diarrhoea but 0.1 g/kg
was safe without causing effects on liver and kidney functions (Kevin
et al., 2012). Aqueous extract studied for acute toxicity in male Wistar
rats using at 1000 and 5000 mg/kg body weight. The ethanolic extract of
leaf causes toxicity doses at higher than 300 mg/kg BW can produce
signs of biochemical and histopathological toxicity in liver, kidney and
heart in wistar albino rats. The dose of 2000 mg did not cause mortality.
SGOT, SGPT, creatinine phosphokinase, LDH, urea and creatinine were
increased in 300 mg/kg BW and 2000 mg/kg BW doses (Vutukuri et al.,
2017). Therefore, ethanolic methanolic and aqueous aqueous extracts of
leaves found to be safety in dose dependent manner.
The study recommends the use of lower concentrations than 1000
mg/kg in order to increase the safety margin. Safety measures that
include monitoring of the vital serum enzyme and hematological pa­
rameters are recommended when this extract is being administered.
Comprehensive screening for possible toxicity on sub-acute, sub-chronic
and chronic levels was also recommended (Kabubii et al., 2015). A study
has been reported the poisoning effects of C. roseus in sheep during long
time grazing of this plant. This study demonstrates that C. roseus can
cause toxicosis in sheep resulting in mortality probably due to dissem­
inated intravascular coagulation and circulatory disturbances related to
diarrhoea and dehydration. In conclusion, results of this study suggested
that the diagnosis of this poisoning depends upon a combination of
anamnesis, clinical signs, laboratory parameters and pathological ex­
amination (Aydogan et al., 2015). The acetone extract of root has been
affected on the larval mortality, fecundity and fertility of Spodoptera
litura (Fabricius) as concentration increases. The maximum mortality
37
Journal of Ethnopharmacology 284 (2022) 114647
was observed in the concentration of 5% as 76.67 ± 21.34% and in the
control there was no mortality (Kumar et al., 2011).
Bisindole alkaloids have been used in clinic applications due to their
antitumor effects. Toxicities of bisindole alkaloids are significantly
different despite similar structures. However, the structure-toxicity re­
lationships of bisindole alkaloids remain unclear. Wang et al. (2017)
have evaluated the toxic effects of bisindole alkaloids using Tetrahymena
thermophile BF5 (T.t. BF5) which could dynamically monitor the growth
of T.t. BF5. The IC10 results showed the relative toxicity of bisindole
alkaloids was compound 112 (105.0 ± 6.3 μg/mL) > compound 121
(135.1 ± 5.1 μg/mL) > compound 111 (215.5 ± 4.7 μg/mL) > com­
pound 143 (314.2 ± 5.9 μg/mL). Structure-toxicity relationships anal­
ysis of these alkaloids indicated that the acylamide at C-3 position and
hydroxyl at C-4 position might contribute to more toxicity. A long
conjugation effect appearing in bisindole alkaloids could increase the
toxicity when the N1 position was substituted with the formyl group.
In humans, the dose-limiting toxicity of compound 112 is a periph­
eral neuropathy. The effects are believed to be limited to peripheral
nerves because compound112 does not penetrate the blood–brain bar­
rier. Compound 112 was interrupts microtubular function, resulting in
an inability to maintain axonal membrane integrity (Grush and Morgan,
1979; Bradfield et al., 2015; Li et al., 2020). Compound 111 is typically
well tolerated, with rare GI or myelosuppressive side effects. Extrava­
sation of compound 111 may cause significant tissue irritation and
cellulitis; therefore the drug must be given through a clean IV puncture
(King and Boder, 1979).
The ethnobotanical surveys did not mention any toxicity associated
with the use of this plant. Still, there may be inadequate evidence to
overrule the toxicity and safety of this plant. Hence, further in-depth in
vitro and in vivo studies for toxicity must be considered.
9. Conclusion
This communication provides an updated and critically analyzed
summary concerning the botany, traditional uses, phytochemistry,
separation, analysis techniques and pharmacology and toxicity aspects
of parts of C. roseus. Traditional uses of C. roseus are confirmed by
different studies pertaining to pharmacological functions such as anti­
microbial, antifertility effect, antioxidant, antidiabetic, hep­
atoprotective, larvicidal/pupicidal effect in different extracts especially
alcoholic extracts of leaves and its fractions. The phytochemical in­
vestigations revealed that bisindole and MIAs were the two major classes
of phytoconstituents that contribute to the various biological effects and
localized in leaves. The phenolic acids and flavonoids are responsible for
antioxidant properties whereas antimicrobial activity could be due to
presence of terpenes, and volatile compounds which are reported as a
minor class of compounds. The recent clinical studies of the most active
phytochemicals, compounds 111, 112 and 121 are employed for the
treatment of various types of chemotherapy due to their cell toxicity.
The toxicological researches of C. roseus are expected to provide the
important data which may contribute to drug discovery having
improved and intriguing pharmacology aspects. We hope these sum­
marized data will be a prevalent aspect of future drug discovery
research.
CRediT authorship contribution statement
Sunil Kumar: conceived the idea, designed, collected, compiled, and
prepared the manuscript. and, wrote the article, prepared tables., drew
the chemical structures, designed the graphical abstract, using Chem­
Draw pro software, and cited the references as per JEP guidelines, hel­
ped in editing the manuscript. Bikarma Singh: conceived the idea,
designed, collected, compiled, and prepared the manuscript, helped in
editing the manuscript. Ramesh Singh: helped in editing the
manuscript.
S. Kumar et al. Journal of Ethnopharmacology 284 (2022) 114647

Declaration of competing interest Hybrid Triple Quadrupole-Linear Ion Trap Mass Spectrometry
UPLC Ultra-Performance Liquid Chromatography
The authors declare no conflict of interest. UV–vis UltraViolate Visible
VLDL-c Very Low Density Lipoprotein
Acknowledgments XRD X-Ray Diffraction Patterns

Authors are thankful to Principal, Ma. Kanshiram Government De­ References

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