TECHNIQUES IN PHYTOCHEMICAL STUDIES

PCG 301

Igoh Aisha Oyiwoja - Bhu/20/01/05/0011

Abdul shola Chris - Bhu/19/01/05/0077
Ovorumu Shalom jemimah - Bhu/19/01/05/0018
Balogun Ayomikun Blessing - Bhu/19/01/05/0022
Boladale Kosisochukwu Dehinsilu - Bhu/19/01/05/0084
Offiong, Emmanuella John - Bhu/19/01/05/0098
Emmanuel ayenajeyi zipporah - Bhu/19/01/05/0071
Kakaan vershima - Bhu/19/01/05/0035
Jones Maureen - Bhu/19/01/05/0020
Chikelue Joshua - Bhu/19/01/05/0123
Dominic Dominique - Bhu/19/01/05/0094
Egwu Victor Enyojo - Bhu/19/01/05/0004
Ngwoke Winifred Nneka - Bhu/19/01/08/0033
Abuke Samuel - Bhu/19/01/05/0066

INDOLE ALKALOIDS
Introduction
Biological source
Phytochemistry
Biosynthesis
Pharmacology
Medicinal uses
Toxicity

INDOLE ALKALOIDS

Indole (1-H-indole) is a benzopyrrole in which the benzene and pyrrole rings are through the
2, 3-positions of the pyrrole. The indole nucleus is found in a large number of naturally
occurring compounds. It is of commercial importance as a component of perfumes.
Isoindole (I-Hisoindole), the isomer in which the benzene and pyrrole rings are fused
through the 3- and 4-positions of the
pyrrole, is not stable. A few of its derivatives are known,
the simplest being N-methylisoindole Indole was first obtained (and its structure elucidated)
in 1866 by Adolf von Baeyer. Interest in indole chemistry
revived about 1930 when it was discovered that the essential amino acid, tryptophan, the
plant growth hormone, heteroauxin, and several groups of important alkaloids are indole
derivatives. It was shown that 3-methylindole (skatole) is produced with indole during
pancreatic digestion or putrefactive decomposition of proteins and, hence, both are found in
the intestines and feces. Interest has centered on medicinal and biochemical aspects of indole
chemistry. Serotonin, which has been identified as a metabolite in brain chemistry; the
psychotomimetic indoles, psilocin and psilocybin from mushrooms; the tranquilizer
reserpine; and the melanin pigments are a few of the compounds that have been studied.
Indole is a colourless crystalline solid (mp 52–54°C,
bp 254°C). The heat of combustion at constant volume is 4,268 MJ/mol (10–20 kcal/mol).
The molecule is planar and has only moderate polarity. Indole has good solubility in a wide
range of solvents including petroleum ether, benzene,
chloroform and hot water. The solubility in cold water is only 1:540 at 25°C; thus, water is a
good solvent for purification by recrystallization. Indole forms salts with high concentrations
of both strong bases and strong acids.
The various plants containing indole alkaloids are vinca,
ergot, Rauwolfia, nux vomica, physostigma, etc.

ERGOT
Synonyms
Ergot; Rye Ergot; Secale cornutum; Spurred rye; Ergot of rye; Ergota.

RAUWOLFIA
Synonyms
Sarpagandha, Chandrika; Chootachand; Indian snake root

NUX VOMICA
Synonyms

Semen strychni, Nux vomica Seed, Poison Nut, Semen strychnos, Quaker Buttons,
Bachelor’s buttons, Dog buttons, Vomit nut, Crow fig.

VINCA
Synonyms
Vinca rosea, Catharanthus, Madagascar periwinkle. Barmasi.

PHYSOSTIGMA
Synonyms
Calabar bean, Ordeal bean, Chop nut.

Peganum harmala contains monoamine oxidase-inhibiting β-carboline alkaloids

 

 Physostigma venenosum "Calabar bean", source of physostigmine (coloured plate
from Köhler's Medicinal Plants)
 

Biological Source
Ergot
Ergot is the dried sclerotium of a fungus, Claviceps purpurea Tulasne, belonging to family
Clavicipitaceae, developing in the ovary of rye plant, Secale cereale (Family Poaceae). Ergot
should yield about 0.15% of the total alkaloids calculated as ergotoxine and water-soluble
alkaloids equivalent to about 0.01% of ergonovine.
Rauwolfia
Rauwolfia consists of dried roots of Rauwolfia serpentina Benth., belonging to family
Apocynaceae.
Nux vomica
Nux vomica consists of the dried ripe seeds of Strychnos nux vomica Linn, belonging to
family Loganiaceae; containing not less than 1.2% strychnine.

Vinca

Vinca is the dried entire plant of Catharanthus roseus Linn.,

belonging to family Apocynacea

Physiostigma
Calabar beans are the dried ripe seeds of Physostigma venenosum half containing not less
than 0.15% alkaloids, belonging to family Leguminosae (Papilionaecae).

Phytochemistry

Ergot
A large number of alkaloids have been isolated from the Ergot. The most important alkaloids
are ergonovine and ergotamine. On the basis of solubility in water the alkaloids are divided
into two groups: water-soluble ergometrine (or ergonovine) group or water-insoluble
(ergotamine and ergotoxine) groups as given hereunder:

Water-soluble group
I. Ergometrine group Ergometrine, Ergometrinine

Water-insoluble group
II. Ergotamine group Ergotamine, Ergotaminine, Ergosine,

Ergosinine
III. Ergotoxine group Ergocristine, Ergocristinine, Ergocryptine,

Ergocryptinine, Ergocornine, ErtgocorninOnly the first group, ergometrine group, belongs to

water-soluble compounds. Alkaloids of Group II and III are polypeptides in which lysergic
acid or isolysergic acid is linked to amino acids. Alkaloids obtained from lysergic acid are
physiologically active compounds. In the first group, for example, ergometrine alkaloids,
lysergic acid or its isomer is linked to an amino alcohol.
The ergot alkaloids (ergolines) can also be divided into two classes (1) the clavine-type
alkaloids, which are derivatives of 6,8-dimethyl-ergoline and (2) the lysergic acid derivatives,
which are peptide alkaloids and contains the pharmacologically active alkaloids that
characterize the
ergot sclerotium (ergot). Each active alkaloid occurs with an inactive isomer involving
isolysergic acid.
Rauwolfia
Rauwolfia contains about 0.7–2.4% total alkaloidal bases from which more than 80 alkaloids
have been isolated. The prominent alkaloids isolated from the drug are reserpine,
rescinnamine, ψ-reserpine, rescidine, raubescine and deserpidine. The other alkaloidal
components are ajmalinine, ajmaline, ajmalicine (8-yohimbine), serpentine, serpentinine,
tetrahydroreserpine, raubasine, reserpinine, isoajamaline and yohambinine.
The other substances present are phytosterols, fatty acids, unsaturated alcohols and sugars.
Nux vomica
Nux vomica contains the alkaloids, Strychnine (1.25%) and
Brucine (1.5%), also traces of strychnicine, and a glucoside Loganin, about 3% fatty matter,
caffeotannic acid and a trace of copper. It contains about 2.5–3.5% bitter indole alkaloids.
Strychnine is therapeutically active and toxic alkaloid and is located in central portion of
endosperm. Brucine is chemically dimethoxystrychnine and is less toxic and has very little
physiological action. It is intensely bitter and is used as a standard for determining the bitter
value, of many bitter drugs. Brucine is more in the outer part. Vomicine and
pseudostrychnine are minor alkaloids.The seeds also contain chlorogenic acid or caffeotannic
acid. Alkaloids are combined with chlorogenic acid or caffeotannic acid. Loganin, a
glucoside is also present. Cell walls of endosperm of nux vomica are thick walled and contain
reserve material hemicellulose consisting of mannan and galactan which on hydrolysis yield
mannose and galactose. Fatty matter is 3% aleurone grains and a trace of copper is present in
the endosperm of the seed. The pulp of the fruit contains about 5% of loganin together with
the alkaloid strychnicine.

Vinca

Alkaloids are present in entire shrub but leaves and roots contain more alkaloids. About 90
alkaloids have been isolated from Vinca from which some like Ajmalicine, Serpentine and
Tetrahydroalstonine are known and are present in other species of Apocynaceae. The
important alkaloids in Catharanthus are the dimer indole indoline alkaloids Vinblastine and
Vincristine and they possess definite anticancer activity. Vindoline and Catharanthine are
indole monomeric alkaloids. It also contains monoterpenes, sesquiterpene, indole and
indoline glycoside

Physiostigma

Drug contains 0.1–0.2% indole alkaloids of which half is physostigmine known also as
eserine (a crystalline solid, white or pinkish coloured, readily soluble in alcohol, sparingly
soluble in water) and is the important alkaloid. The other alkaloids are eseramine, geneserine
and physovenine. Physostigmine the major alkaloid is present in cotyledons up to 0.04–0.3%,
Physostigmine is methyl carbamide acid ester of eroline. These alkaloids are
pyrrolidineindoline derivatives. Calabar beans also contain stigmasterol.

Biosynthesis

The Voacanga Indole Alkaloids

A number of old phytochemical literatures are available for the indole alkaloids of
Voacanga. It has been shown in many species that these secondary metabolites are highly
localized in the different parts of the plant such as the leaves, roots, stem, branch, and
seeds. It has to be noted that earlier studies pertaining to V. dregei alkaloids have been
included under V. thouarsii due to botanical revision. Biosynthetic formation. The
monoterpenoid indole alkaloids are biosynthetically made from tryptophan and
secologanin, a terpene iridoid. It is believed that the corynanthe alkaloids are the precursor
anabolites for the more structurally complex aspidosperma, iboga, and strychnos alkaloid
(15). Preakuammicine (a strychnos-type intermediate and astrictosidine derivative) is the
common precursor of the aspidosperma, strychnos and iboga alkaloids. Stemmadenine
undergoes a rearrangement reaction to afford, dehydrosecodine, an acrylic ester
intermediate, which serves as a common precursor for the aspidosperma and the iboga
structures (16-18). It has not been verified through biosynthetic experiments that the iboga
alkaloid, catharanthine, and the aspidosperma alkaloid, tabersonine, are produced by way
of Diels–Alder reaction of dehydrosecodine

The monoterpenoid indole alkaloids from Voacanga Thouars

Biogenetic precursor of all indole alkaloids is the amino acid tryptophan. For most of them,
the first synthesis step is decarboxylation of tryptophan to form tryptamine.
Dimethyltryptamine (DMT) is formed from tryptamine by methylation with the participation
of coenzyme of S-adenosyl methionine (SAM). Psilocin is produced by spontaneous
dephosphorylation of psilocybin.

In the biosynthesis of serotonin, the intermediate product is not tryptamine but 5-

hydroxytryptophan, which is in turn decarboxylated to form 5-hydroxytryptamine
(serotonin).
Biosynthesis of β-carboline alkaloids occurs through the formation of Schiff base from
tryptamine and aldehyde (or keto acid) and subsequent intramolecular Mannich reaction,
where the C(2) carbon atom of indole serves as a nucleophile. Then, the aromaticity is
restored via the loss of a proton at the C(2) atom. The resulting tetrahydro-β-carboline
skeleton then gradually oxidizes to dihydro-β-carboline and β-carboline. In the formation of
simple β-carboline alkaloids, such as harmine and harmaline, pyruvic acid acts as the keto
acid. In the synthesis of monoterpenoid indole alkaloids, secologanin plays the role of the
aldehyde. Pirroloindole alkaloids are synthesized in living organisms in a similar
way.Biosynthesis of ergot alkaloids begins with the alkylation of tryptophan by dimethylallyl
pyrophosphate (DMAPP), where the carbon atom C(4) in the indole nucleus plays the role of
the nucleophile. The resulting 4-dimethylallyl-L-tryptophan undergoes N-methylation.
Further products of biosynthesis are chanoclavine-I and agroclavine – the latter is
hydroxylated to elymoclavine, which in turn oxidizes into paspalic acid. In the process of
allyl rearrangement, paspalic acid is converted to lysergic acid.
Biosynthesis of monoterpenoid indole alkaloids begins with the Mannich reaction of
tryptamine and secologanin; it yields strictosidine which is converted to 4,21-
dehydrogeissoschizine. Then, the biosynthesis of most alkaloids containing the unperturbed
monoterpenoid part (Corynanthe type) proceeds through cyclization with the formation of
cathenamine and subsequent reduction to ajmalicine in the presence of nicotinamide adenine
dinucleotide phosphate (NADPH). In the biosynthesis of other alkaloids, 4,21-
dehydrogeissoschizine first converts into preakuammicine (an alkaloid of subtype strychnos,
type Corynanthe) which gives rise to other alkaloids of subtype strychnos and of the types
Iboga and Aspidosperma. Bisindole alkaloids vinblastine and vincristine are produced in the
reaction involving catharanthine (alkaloid of type Iboga) and vindolin (type Aspidosperma).

WHAT IS PHARMACOLOGY
Pharmacology is a branch of science that deals with the study of drugs and their actions on
living systems – that is, the study of how drugs work in the body (sometimes referred to as
‘drug actions’). To understand this we need to consider what a drug is, how it affects our
physical, emotional and psychological wellbeing, the type of drug being used, the modes of
administration, how the drug is absorbed and the characteristics of the person taking the drug.
Pharmacology is responsible for painkillers, caffeine drinks and antibiotics. Without
pharmacologists we wouldn’t be able to:
Discover new medicines to help fight diseases
Improve their effectiveness and reduce unwanted side effects
Understand why people have different responses to medicines, and why some work better for
some people than others
Understand why some drugs cause addiction
Pharmacology has two major branches:
Pharmacokinetics: which refers to the absorption, distribution, metabolism and excretion of
drugs.
Pharmacodynamics: which refers to the molecular, biochemical and physiological effects of
drugs, including drug mechanism of action

PHARMACOLOGICAL EFFECT OF INDOLE ALKALIODS

Indole compounds, related to the metabolism of tryptophan, constitute an extensive family,
and are found in bacteria, plants and animals. Indolic compounds possess significant and
complex physiological roles, and especially indole alkaloids have historically constituted a
class of major importance in the development of new plant derived drugs. The indole alkaloid
alstonine has been identified as the major component of a plant-based remedy, used in
Nigeria to treat mental illnesses by traditional psychiatrists. Although it is certainly difficult
to compare the very concept of mental disorders in different cultures, the traditional use of
alstonine is remarkably compatible with its profile in experimental animals. Even though
alstonine in mice models shows a psychopharmacological profile closer to the newer atypical
antipsychotic agents, it also shows important differences and what seems to be an exclusive
mechanism of action, not entirely clarified at this point. Considering the seemingly unique
mode of action of alstonine and that its traditional use can be viewed as indicative of
bioavailability and safety, this review focuses on the effects of alstonine in the central
nervous system, particularly on its unique profile as an antipsychotic agent. We suggest that a
thorough understanding of traditional medical concepts of health and disease in general and
traditional medical practices in particular, can lead to true innovation in paradigms of drug
action and development. Overall, the study of this unique indole alkaloid may be considered
as another example of the richness of medicinal plants and traditional medical systems in the
discovery of new prototypic drugs.

MEDICINAL USES OF INDOLE ALKALOID

Ergot
Ergot is oxytocic, vasoconstrictor and abortifacient and used to assist delivery and to reduce
post-partum haemorrhage.
Lysergic acid diethylamide (LSD-25), obtained by partial synthesis from lysergic acid, is a
potent specific psychotomimetic. Ergometrine is oxytocic and used in delivery.
It stimulates the tone of uterine muscles and prevents postpartum haemorrhage.
Only ergometrine produces an oxytocic effect, ergotoxine and ergotamine having quite a
different action.

Ergometrine is soluble in water or in dilute alcohol. It is known as ergonovine. Ergotamine

and the semisynthetic dihydroergotamine salts are used as specific analgesics for the
treatment of migraine. Lysergic acid diethylamide (LSD-25), prepared by partial synthesis
from lysergic acid, is a potent specific psychotomimetic.

Rauwolfia
Rauwolfia in used as hypnotic, sedative and antihypertensive.
It is specific for insanity, reduces blood pressure and cures pain due to affections of the
bowels.
It is given in labours to increase uterine contractions and in certain neuropsychiatric
disorders. Ajmaline, which has pharmacological properties similar to those of quinidine, is
marketed in Japan for the treatment of cardiac arrhythmias.
Reserpine is a white or pale buff to slightly yellow, odourless, crystalline powder that darkens
slowly when exposed to light and rapidly when in solution. Reserpine is an antihypertensive
and tranquilizer. Rescinnamine is the methyl reserpate ester of 3,4,5-trimethoxy cinnamic
acid.

Nux vomica
The properties of nux vomica are substantially those of the alkaloid Strychnine. In the
mouth it acts as a bitter, increasing appetite; it stimulates peristalsis, in chronic constipation
due to atony of the bowel it is often combined with cascara and other laxatives with good
effects.
Strychnine, the chief alkaloid constituent of the seeds, also acts as a bitter, increasing the
flow of gastric juice; it is rapidly absorbed as it reaches the intestines after which it exerts its
characteristic effects upon the CNS, the movements of respiration are deepened and
quickened and the heart slowed through excitation of the vagal centre.
Strychnine has a stimulant action on spinal cord and reflex movements are better. It is
considered as nervine and sex tonic.
The senses of smell, touch, hearing and vision are rendered more acute, it improves the
pulse and raises blood pressure and is of great value as a tonic to the circulatory system in
cardiac failure. In toxic doses strychnine causes violent tetanus like convulsions and death
takes place dueto asphyxia and respiratory failure.
Brucine closely resembles strychnine in its action, but is slightly less poisonous; it paralyses
the peripheral motor nerves. It is said that the convulsive action characteristic of strychnine
is absent in brucine almost entirely. It is used in pruritis and as a local anodyne in
inflammations of the external ear.
Nux vomica is also known as vomiting nut but it has no vomiting properties. However
Strychnos potatorum has emetic action.

vinca
Vinblastin is an antitumour alkaloid used in the treatment of Hodgkin’s disease. Vincristine is
a cytotoxic compound and used to treat leukaemia in children.
Vinca is used inherbal practice for its astringent and tonic properties in menorrhagia and in
haemorrhages generally.
In cases of scurvy and for relaxed sore throat and inflamed tonsils, it may also be used as a
gargle. For bleeding piles, it may be applied externally, as well as taken internally. It is also
used in the treatment of diabetes.
physiostigma
Mainly used for diseases of the eye; it causes rapid contraction of the pupil and disturbed
vision. Also used as a stimulant to the unstriped muscles of the intestines in chronic
constipation. Its action on the circulation is to slow the pulse and raise blood pressure; it
depresses the CNS, causing muscular weakness; it has been employed internally for its
depressant action.
Some monoterpenoid indole alkaloids interact with adrenoreceptors. For example ajmalicin is
a selective antagonist of alpha 1 adrenergic receptors and therefore has antihypertensive
action.
I

TOXICITY OF INDOLE ALKALOIDS

The toxicity profile of indole alkaloids from leaves of A. scholaris was

investigated. In acute toxicity tests, mice were administered total alkaloids (TA)
and five indole alkaloids. In a chronic toxicity test, rats were continuously
administered TA (50, 100, and 300 mg/kg bw) for 13 weeks, followed by a 4-
week recovery. A single administration of TA affected the behaviour of mice,
and at 12.8 g/kg bw, prone position, shortness of breath, wheezing, and
convulsion were observed. The half-lethal dose (LD50) in mice was 5.48 g/kg
bw, almost 2740 times the clinical dose in humans. Among the five indole
alkaloids, the maximum tolerance dose in mice ranged from 0.75 to 4 g/kg bw.
The TA-treated rats did not die and showed no adverse effects or dose-
dependent changes in weight or food and water consumption, despite
fluctuations in haematological and biochemical parameters compared with
historical data. Furthermore, both gross and histopathological observations
revealed no abnormalities in any organ. With daily oral administration to rats,
the non-observed-adverse-effect-level of TA was 100 mg/kg bw. The results
indicate that TA is safe for clinical use.
Traditional medicine has a long history of improving health worldwide, and
thus, these medicines are considered to be a valuable resource for the
discovery of new drugs. In recent years, the number of researchers focusing on
herbal remedies has increased tremendously worldwide in order to verify or
prove their efficacy after long histories of folk use. However, some products
isolated from these herbal medicines have produced adverse effects. For
example, aristolochic acid [1], contained in plants of Aristolochia and
Asarumcan, is well known to cause kidney failure and cancer, particularly of
the urinary tract in humans. Such adverse effects have aroused concern about
the safety of herbal medicines [2]. Furthermore, many nonclinical studies
report adverse effects or toxicities of herbal medicines after long-term use [3,
4]. Therefore, the toxicity of medicinal herbs needs to be critically assessed in
order for them to be used safely by patients and clinicians.

Alstonia scholaris (L.) R. Br. is widely distributed in deciduous and evergreen

forests and even on the plains in the tropical regions of Africa and Asia [5]. The
leaves have long been used in “dai” ethno-pharmacy to treat postinfectious
cough, chronic bronchitis, asthma, and other respiratory tract infections in
Yunnan Province, China [6]. The authors of this paper have investigated
intensively the phytochemical constituents of the different parts of the plant
[7–26]. The chemical profile and metabolites of alkaloidal extract of leaves of
A. scholaris indicate that scholaricine, 19-epischolaricine, vallesamine, and
picrinine are the major indole alkaloids [27, 28].

Moreover, the extract and alkaloids of A. scholaris leaves have antitussive,

anti-asthmatic, expectorant [29], analgesic, anti-inflammatory [30], anti-airway
inflammation [31], and anti-allergic effects [32] and provide protection against
postinfectious cough in vivo [33]. The alkaloids also trigger the activation of β2
adrenergic receptors [34] and inhibit nuclear factor-κB bioactivities in vitro
[35]. Although the authors have reported on the pharmacological effects of A.
scholaris leaves, their in vivo safety remains undetermined. Therefore, the
primary purpose of this paper was to investigate the acute and chronic toxicity
of the indole alkaloids of A. scholaris in mice and rats to help develop
parameters for rational drug use.

Echitamine, a monoterpene indole alkaloid, is isolated from the stem bark of A.

scholaris. Baliga et al. reported that the hydro-alcoholic extract of the bark of
A. scholaris caused mortality and deformity in various organs at the dose of
240 mg/kg bw (30 days) when distributed in India, which were assumed to
result from the contribution of echitamine toxicity [36]. Thus, the acute toxicity
test of echitamine was also investigated in the study which suggested its safety
in acute toxicity evaluation.

Results

Acute Toxicity

In mice in the 12.8 g/kg bw dose group, the TA produced the side effects of
convulsions and death within 7–30 min after oral administration. In the 9.0
g/kg bw group, the symptoms occurred within 5–15 min and included prone
position, shortness of breath, wheezing, convulsions, and death. In the other
groups, the toxic effects were the same but occurred over 1–3 h, and a dose–
response relationship was observed

1). The dead mice were immediately dissected, and general observation
revealed no changes in the size, color, and texture of organs. The collective
findings were used to calculate an LD50 of 5.48 g/kg bw in mice, which is 2740
times the recommended clinical dose.

Acute toxic symptoms of mice after the oral administration of TA

In the Pic group at the dose of 2.0 g/kg bw, the activity of all mice decreased at
1 h after administration. Some animals had shortness of breath and an
unstable gait. Two mice had convulsions and limb vasodilation at 3 h after
administration but recovered 30 min later. These observations indicated that
the maximal tolerated dose (MTD) did not exceed 2.0 g/kg bw in both sexes.

In the Val group at the dose of 4.0 g/kg bw, toxic effects were not observed
during the treatment and observation periods. Thus, the MTD exceed 4.0 g/kg
bw in both sexes.

In the Sch group at the dose of 0.75 g/kg bw, the activity of all mice was
reduced after administration. Moreover, five mice displayed symptoms of
poisoning, such as shortness of breath, unsteady gait, tremors, convulsions,
and death. Therefore the MTD was less than 0.75 g/kg bw in both sexes.
In the Epi and Ech groups at the dose of 2.0 g/kg bw, no mortality or changes
occurred in mice during the treatment and observation periods. Although
reduced activity was observed following administration, the mice recovered 4
h later. Therefore, the MTD was 2.0 g/kg bw in both sexes.

General Observations Associated with Chronic Oral Toxicity

The rats in each group moved freely, had shiny fur, and no deaths occurred
during the study. At the 10th week of administration, sporadic alopecia was
observed in one female rat in the 50 mg/kg bw group, but new hair grew
gradually after ceasing the TA treatment. However, no substantial difference
was detected between control and TA groups. Therefore, the alopecia in the
female rat was presumed to be unrelated to the treatment with TA. No other
abnormal clinical manifestations were observed.

The use of natural drugs is increasingly popular in healthcare and other areas
worldwide. However, their safety remains a major public health problem.
Because these agents are generally perceived as being harmless to the body,
they are used in self-therapy without supervision. Although the bioactive
compounds in medicinal plants exert a variety of beneficial biological effects in
humans, knowledge of their potential toxicities is limited. The leaves of A.
scholaris have a long history of treating whooping cough in the Dai people in
Yunnan Province. Previously, the author’s group demonstrated the efficacy of
the total indole alkaloid extract of A. scholaris leaves in respiratory
management. Although the safety of the extract of stem bark of A. scholaris
has been evaluated [36], the potential toxic effects of the indole alkaloids of
leaves remain unknown. Accordingly, in the present study, the acute and
chronic oral toxicity of the indole alkaloids in the leaves of A. scholaris was
evaluated in rodents.

An acute toxicity test is used to evaluate any adverse effects appearing within
a short time after a single large dose of the test substance or after multiple
doses given within 24 h. In this study, oral exposure to the TA extract at a dose
from 2.2 to 12.8 g/kg bw triggered important toxic responses in mice, including
prone position, tachypnea, whoop, and convulsions, in a dose–response
relationship. These toxic symptoms might be related to toxicity to the
neuromuscular, central nervous system and autonomic nervous system. The
LD50 value for the mice was 5.48 g/kg bw, which is approximately 2740 times
higher than the recommended clinical dosage for patients (2 mg/kg/d). And
the MTD was 2.2 g/kg bw, correspondingly, the doses converted into four main
compounds were 220 mg/kg bw (Pic), 132 mg/kg bw (Sch), 132 mg/kg bw (Val)
and 22 mg/kg bw (Epi), respectively. Of the four compounds tested, both Pic
(2.0 g/kg bw) and Sch (0.75 g/kg bw) caused the same toxic responses of
shortness of breath, unsteady gait, tremors, convulsions, and death. However,
a toxic response was not observed in the Val and Epi groups. Under the
conditions of this experiment, the MTDs were 2.0 g/kg bw (Pic), less than 0.75
g/kg bw (Sch), more than 4.0 g/kg bw (Val) and 2.0 g/kg bw (Epi). In the
analysis of gross anatomy, no abnormalities were observed in any of the
internal organs, including the liver and kidney, when compared with the
control group. The conclusion was that the MTDs of four compounds in TA was
smaller after comparing the tested result with that calculated. We speculated
that there were other trace components and non-alkaloid constituents which
affect the safe dose of total alkaloid extract in addition to the four major
components. Next, the absorption of TA in vivo could be affected by
gastrointestinal enzymes of animals, acid dissociation constant, partition
coefficient (log p), absorption site (stomach-duodenum), chemical and
physiological polymorphisms, cytochromes, etc.

Echitamine, a monoterpene indole alkaloid, did not cause a toxic reaction in

the mice in this study and was well tolerated at the dose of 2 g/kg bw without
any acute toxicity. Furthermore, echitamine has not been detected in the
leaves of A. scholaris collected in Yunnan Province, although it was isolated
from the bark in a previous investigation [37]. Thus, the good tolerance and
the absence of echitamine in all tested leaves of A. scholaris suggested that no
need to limit its quantity in a botanic drug from TA.

A chronic toxicity study is typically conducted from 1 to 3 months, because

some substances that do not cause immediate toxicity may cause toxic effects
after repeated exposure. According to the testing guidelines for the safety
evaluation of drugs (Notification [Z] GPT3-1) issued by the China Food and Drug
Administration on March 2005, the period for testing drug administration in
animals should be based on the expected period of clinical use of that
substance in humans. The repeated oral administration of a test substance for
1–3 months in animals is thought to be comparable to the administration for
less than 1 month in humans. The objective of chronic toxicity studies is to
determine the possible clinical adverse reactions caused by the substance,
including the nature and degree of harm, the dose–response and time–
response relationships, the effects on target organs or tissues, and the
reversibility, and then predict the starting dose in clinical trials and the safe
dose range for repeated drug use. The doses of 50, 100, and 300 mg/kg bw TA
used in this study were equivalent to 25, 50, and 150 times, respectively, the
usual dose in humans.

In this study, both male and female rats treated with doses of 50, 100, and 300
mg/kg bw TA once daily showed no signs of clinical toxicity or mortality.
Changes in food and water ingestion are generally used as indicators of the
harmful effects of drugs and chemicals [38]. In the experiments in this study,
the two parameters were not significantly affected in weekly measurements,
except for slight fluctuations in the TA treatment groups relative to the control
group that were within the normal ranges of values. In addition, the alopecia
of one female rat in the 50 mg/kg bw group was considered incidental given
the frequencies across groups. Thus, the findings of this study suggested that
different doses of TA from 50 to 300 mg/kg bw intragastrically administered to
rats for 13 weeks had no significant effects on general behavior, mental state,
or food intake.

The liver and kidneys are frequent targets of drug action, because the liver is
the primary organ for drug biotransformation, and the kidneys are the primary
organs for drug excretion. Organ weight is one of the main parameters used to
evaluate the effects of test substances in toxicity studies [39]. In the present
investigation, the liver and kidney organ coefficients in the 300 mg/kg bw
group increased significantly in the 7th and 13th weeks compared with those
in the control group. Meantime, we found the body weight showed a
decreasing trend at the corresponding time. It thus was speculated the
decrease of the two coefficients was induced by the decrease of body weight.
However, weight loss was due to shrinkage of absorption of nutrients by the
intestinal, but the slight fluctuations of food/water consumption in the TA-
treated groups were within the normal ranges of values. In addition, no
significant differences were observed in the serum levels of ALT, AST, CRE and
BUN. Some test substances may harm tissues at the cellular level but not cause
any observable abnormalities in an organ. For this reason, histopathological
examination of livers and kidneys was conducted to identify any cellular
damage in the internal organs or tissues. Fortunately, no significant
histopathological changes in liver tissues in the 300 mg/kg bw group were
observed relative to the control group. Moreover, the biochemical indicators
of liver and kidney function were normal after the withdrawal of TA for 4
weeks. The above results all indicated that the increase in liver and kidney
coefficients during the administration period was not toxicologically significant.
In addition, Treatment-related changes of Glu, an indicator of pancreatic
function [40], was not observed, signifying that the TA did not damage
pancreas function.

The hematopoietic system, which is highly sensitive to toxic substances, can be

altered when poisonous plants are ingested [41]. And changes in hematological
parameters can reflect adverse effects on bone marrow function, such as
anemia and hemolysis [42]. White blood cell counts are commonly determined
to assess immune function [40]. In this study, white blood cell and red blood
cell counts of the TA-treated groups were comparable to those of the control
group. In addition, no significant differences in weight of the thymus were
observed, an important lymphoid organ associated with the immune system
[43]. The exception was the higher WBC counts in the 300 mg/kg bw male and
female rats than those in the controls at the 7th and 13th weeks. The values of
the other hematological parameters examined (Hb, RDW, MCV, MCH, PLT, PT,
and RET) were within the ranges of normal reference values [39]. Thus, the
collective findings of the study indicated no adverse effects of TA on the
hematology and immune system of rats.

Histopathological examination is a fundamental step in preclinical toxicology

research that is used to further validate whether tissues or internal organs are
damaged [42]. According to the Organization for Economic Co-operation and
Development guidelines, microscopic examination of organs is not required in
the 50 mg/kg bw group in cases in which no histopathological alterations are
observed in the 300 mg/kg bw group [44]. In this study, no apparent
histopathological changes were observed in the main organs and tissues
(heart, kidney, spleen, stomach, small intestine, colon, brain, adrenal gland,
testis, ovary, uterus) of rats treated with 300 mg/kg bw TA. The exceptions,
which were observed in both the control and 300 mg/kg bw groups, were small
focal inflammations in livers, local interstitial pneumonia in lungs, and
interstitial lymphocyte infiltration in prostates. According to previous reports
[45], these types of lesions are commonly associated with Sprague–Dawley
rats of the age used in this study and are considered to be related to
spontaneous or iatrogenic causes. The lesions were similar in severity in all
groups and were graded as either minimal or mild and were therefore not
considered as TA-related. Thus, the test substance showed no accumulative
toxicity when administered at doses of 50, 100, and 300 mg/kg bw TA for
consecutive 13 weeks and after a 4-week recovery period following withdrawal
of the TA treatment. All these results indicated that the TA and the five main
compounds were safe for human use.

Conclusions

In summary, at the tested oral doses ranging from 2.2 to 12.8 g/kg bw, TA
produced some signs of acute toxicity in mice in a dose-dependent manner,
which might be related to toxicity to the neuromuscular, central nervous
system and autonomic nervous system. The half-lethal dose (LD50)
(administered via gavage) in mice of 5.48 g/kg bw was approximately 2740
times the recommended dose for patients. In addition, acute toxicity testing
demonstrated that picrinine, vallesamine, sholaricine, 19-epischolaricine, and
echitamine were safe for mice when used alone. Moreover, no lethality,
significant alterations in hematological and serum biochemical indices, or
adverse histopathological effects were evident in the chronic toxicity study (13
weeks) with rats using doses 25, 50, and 150 times the clinical dose. On the
basis of this result, the TA should be considered safe for use in clinics and
deserve further development as a pharmaceutical product.

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