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Plant

Milk Thistle, Silibum marianum, from the family Asteraceae (daisy’s family), is a flower used for
thousands of years for medical and veterinary purposes.
It is native to the Mediterranean (Southern and Western Europe) but is nowadays spread
throughout the world (mainly South and North America), growing in dry, sunny areas.
Occasionally called St. Mary’s Thistle, Holy Thistle, Marian Thistle, Mary Thistle, Lady’s Thistle,
Sow Thistle, Thistle of the Blessed Virgin, Christ’s Crown, Venus Thistle, Heal Thistle,
Variegated Thistle, or Wild Artichoke, it has stem branches at its top, reaching a height of 4-10
feet (1,22-3,05m). It has red-purple flowers and a small, hard-skinned, brown, spotted, and
shiny fruit.

Description
Milk Thistle’s fruit and seeds contain a lipophilic mixture of biologically active flavonolignans
known as silymarin. This complex comprises 2-3% of the dried herb and is composed by three
primary isomers: silybin (also known as silibinin or silybinin), silychristin (silichristin) and
silydianin (silidianin). Silybin is the most prevalent and biologically potent of the three isomers
and accounts for 50-70% of the silymarin complex, being accounted responsible for the
benefits attributed to silymarin. Silymarin also contains sterols, fixed oil, flavonoids (apigenin,
quercetin, kaempferol), lignans, biogenic amines (tyramine, betaine) and mucilage.

History
Most of the names attributed to Milk Thistle reflect the legend that compares the white leaf
veins to a drop of the Virgin Mary’s milk and, thus, the location of the hidden baby Jesus
during the family’s flight from Egypt.
This plant has been since the Greeks and the Romans as an herbal remedy for a variety of
ailments, especially liver disorders. Later, in the 16th and 17th centuries, it was mentioned in
many of the great herbals, where it was recommended for melancholy (liver disorder).
In the late 19th and early 20th centuries, it was introduced in the Eclectic practice after 1898,
with specific indications, as follows: “splenic, hepatic and renal congestion, face swallow,
appetite, capricious; nervous irritability; dependency; physical debility; pain in either
hypochondriac; pelvis tension and weight; congestion of the parts supplied by the coeliac axis;
and non-malarial splenic hypertrophy”.
Clinical Studies
• “The hepatoprotective effects of silymarin (and silybin, an isomer of silymarin) have
been shown in several in vitro studies as well as studies in animals, including dogs,
with induced liver damage.”
• “Studies in dogs with carbon tetrachloride toxicity and dogs with phalloidin toxicity
showed that silymarin was protective Silymarin, thus far, has shown to be safe.
• In one placebo-controlled experimental study of dogs poisoned with the Amanita
phalloides mushroom, silybin had a positive effect on liver damage and survival
outcome.”
• “In two trials of dogs given hepatotoxic chemicals, silymarin or silibinin improved
biochemical and histologic measures of hepatotoxicity, and survival was improved. In
the first trial, beagles were given 85mg/kg Amanita phalloides lyophilizate orally. In
addition to a control group, 4 groups of 6 to 10 dogs were given the following:
▪ Prednisolone, 30mg/kg IV at 5 and 24 hours
▪ Cytochrome C, 50mg/kg IV at 5 and 24 hours
▪ Penicillin G, 1000mg/kg IV at 5 hours
▪ Silymarin, 50mg/kg IV at 5 hours and 30mg/kg IV at 24 hours
o Blood was sampled at 5, 24, 48, 96, and 192 hours. Results of this study
showed that all liver enzymes of dogs that received milk thistle remained
nearly normal throughout the testing period, whereas those of control dogs
increased precipitously (Floersheim, 1978). Vogel (1984) administered
85mg/kg of amanita lysate to beagles, then treated half of them with silibinin
(78mg/kg IV at 5 and 24 hours). In the control group, 4 of 12 dogs died, and
histopathology showed severe liver necrosis. None died in the silibinin-treated
group, and liver histopathology was nearly normal.”

Mechanisms of Action
• Liver
o Inhibition of lipid peroxidase and β-glucuronidase;
o Hepatoprotective function through:
▪ Increased hepatic regeneration (by stimulating RNA polymerase,
ribosomal RNA - therefore increasing protein synthesis and increasing
gene transcription and translation -, and potentially DNA synthesis);
▪ Scavenging oxygen radicals, hence promoting an antioxidant effect;
▪ “Stabilization” of hepatocyte cell membranes, thus preventing toxin
penetration by controlling its permeability;
▪ Competitive inhibition of toxins that might otherwise bind to hepatic
and damaged cell membrane receptors (ex.: Amanita phalloides);
o Anti-inflammatory effects reflected by the inhibition of lipoxygenase’s (5-LOX)
pathway and subsequent leukotriene synthesis, as well as mast cells
stabilization, suppression of hydrogen peroxide and superoxide, and inhibition
of nuclear factor kappa (NF-ƙB);
o Anti-fibrotic qualities, through inhibition of stellate cell activation and
proliferation, as well as signaling for type I collagen synthesis and production
of metalloproteinase I tissue inhibitor;
o Modulation of hepatocyte transport (P-glycoprotein), which is important in the
liver’s ability to promote choleresis, therefore increasing bile flow through the
expansion of the endogenous pool of bile salts, including the hepatoprotective
bile acid ursodeoxycholic acid;
 o Inhibition of several liver enzymes, including cytochrome P450 (CYP450),
CYP3A4-, and CYP2C9-, which are enzymes involved in drug metabolism –
because many toxins require activation by CYP enzymes, any compound that
inhibits cytochrome enzymes will reduce or eliminate the toxicity of
hepatotoxins;
o Exerts a decrease in low-density lipoprotein (LDL, “bad”) cholesterol, a
decrease in liver cholesterol concentration and a decreased level of serum
cholesterol, possibly by modulating the absorption of cholesterol;
o Anti-cirrhotic effect by inhibition of the transformation of hepatocytes into
fibroblasts;
o Heavy metal chelation, including iron and decrease in glutathione destruction
in iron overload conditions;
• Chemoprotection
o Inhibition of the cytotoxic, inflammatory, and apoptotic effects of tumor
necrosis factor (TNF);
o Inhibition of the intestinal β-glucuronidases, modulating carcinogen
metabolizing enzymes and inhibiting epidermal growth factor receptor and
kinases;
o Prevention of cancer development through alteration of the cell cycle
progression, inhibition of cell survival signaling and mitogenic signaling,
synergizing the effects of doxorubicin, inhibition of the secretion of
proangiogenic factor, inhibition of cell growth and enhancing cell apoptosis;
• Improvement of glycemic control in type 2 diabetes;
• Reduction of oxidative damage to kidney cells;
• Reduction of damage to the pancreas, for example from cyclosporine toxicity;
• Decrease in calcium metabolism.

Pharmacokinetics
Even though all parts of Milk Thistle have been used medicinally throughout History, the
concentration of silybin is highest in the seeds and leaves, from which the flavonoids are
extracted.
Extract preparations with silymarin are standardized to contain 70-80% flavonolignans, as this
concentration is considered necessary for effective absorption of the products.
Silybin can be administrated either per os (PO) or intravenously (IV), and it is poorly soluble to
insoluble in water, which confers it a low oral uptake and bioavailability. However, there is
evidence that its absorption is significantly increased when complexed with
phosphatidylcholine, therefore leading to a lower dose in formulations.
Silybin reaches much higher concentrations in bile and in liver cells than in serum, which
indicates that this drug undergoes extensive enterohepatic circulation. Its elimination is
primarily via bile as glucuronide or sulfate conjugates, therefore, a total of an oral dose can be
found 20-40% in bile, while only 3-8% is excreted in the urine.
Silybin levels are known to peak in bile at 2 and 9 hours post ingestion, and excretion in the
bile continues for 24h. As for cats, its half-life is 3.2 hours (+/- 1.74).
There is scientific evidence that demonstrates that its distribution occurs mostly in the organs
of the digestive tract, such as the liver, pancreas, and the stomach, but can be also found in
the lungs.
This is a safe and well tolerated nutraceutical, with a documented 50% lethal dose (LD50) of
140mg/kg for dogs IV and 10g/kg PO, with a maximum tolerated dose of 300mg/kg.

Silibinin-Phospholipids Complex
The goal of any drug delivery system is to improve bioavailability by preventing premature
degradation and enhancing drug uptake. This is achieved through maintaining drug
concentration within the therapeutic threshold by controlling the drug release rate, i.e.,
improve the spatial placement and temporal delivery of the drugs. Therefore, it comprises the
reduction of the side effects by effective and more precise targeting to the disease site and
target cells.
Lipid solubility and molecular size are the major limiting factors for molecules to penetrate the
biological membranes, in order to be absorbed systematically, following oral or topical
administration.
The effectiveness of any product (whether medication or herbal) is dependent upon delivering
an effective level of active compounds. However, some herbal phytomolecules are poorly
miscible with oils and other lipids, thus often failing to penetrate through the small intestine
because of its lipoidal nature.
Silibinin is poorly water-soluble (solubility <50 μg/mL) because of its highly hydrophobic and
nonionizable structure. This causes both its inefficient absorption in the intestine and its
elevated metabolism in the first liver passage after its absorption, two mechanisms that
decrease hematic concentration and, consequently, the arrival at the target organ.
PHYSICOCHEMICAL PROPERTIES OF Value
SILYMARIN
Molecular formula C25H22O10
Molecular weight (g mol−1) 482.44
Solubility 49.7 g· ml−1 (pH 7.4 PBS)
Melting point 156 ºC
max of absorbance 288 nm
LogP 2.8
In order to overcome these aspects that define silibinin, there have been extensive attempts to
enhance its bioavailability (by dissolution velocity and solubility increase), such as the synthesis
of a silibinin-phospholipid complex, proven to be an optimal method to potentiate silibinin’s
therapeutic action.
In a specific study aimed at enhancing silibinin bioavailability, the researchers described a new
protocol to prepare a silibinin-phospholipid complex. Solubility studies confirmed the higher
solubility of silibinin-phospholipid complex in water (78.25±2.68 μg/mL) and n-octanol
(62.67±1.86 mg/mL). Following an oral administration of 9.1 mg/kg of silibinin-phospholipid
complex and silibinin-N-methylglucamine to rats, there was a significant increase in the
bioavailability of silibinin after the oral administration of silibinin-phospholipid complex
compared to silibinin-N-methylglucamine.

The Complex
Phospholipids, the major constituents of cell membrane, are amphiphilic molecules with
solubility in aqueous and oily mediums, which contain a hydrophilic head and a hydrophobic
tail.
Two types of phospholipids are distinguished: glycerophospholipids such as
phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine,
cardiolipin and derivatives of glycerol and sphingophospholipids (which do not contain
glycerol) such as sphingomyelin.
These substances are readily compatible with the entire range of vitamins, minerals,
metabolites, and herbal preparations, thus being capable to form complexes with silybin for
improving its bioavailability and the therapeutic efficacy.
The major advantages of phospholipids-based carrier system in comparison to other delivery
systems are:
• Enhanced permeation of drug through skin for transdermal and dermal delivery;
• Function as platform for the delivery of a large and diverse group of drugs (peptides,
protein molecules);
• Their composition is safe, and the components are approved for use in animals;
• Low risk profile- the toxicological profiles of the phospholipids are well documented in
the scientific literature;
• High market attractiveness for products with proprietary technology;
• Relatively simple to manufacture with no complicated technical investments required
for its production;
• The vesicular system is passive, non-invasive and is available for immediate
commercialization.

Phosphatidylcholine (PC), the mostly used phospholipid in Phytosomes with silibinin, is the
most abundant phospholipid of all mammalian cells and subcellular organelles. PC comprises
40–50% of total cellular phospholipids, although different cell types, individual organelles, and
even the two leaflets of organelle membranes contain distinct phospholipid compositions.
Phytosome (“Phyto”, plant, and “Some” cell-like), is a technique applied in
phytopharmaceutical products for the enhancement of bioavailability of herbal extracts for
medicinal applications. This technology enables the delivery of herbal products and drugs with
improved absorption and, as a result, producing better pharmacokinetic and
pharmacodynamic profiles than those obtained by conventional herbal extracts, without
resorting to pharmacological adjuvants or structural modification of the ingredients.
Phytosomes are developed when the standardized extract and active ingredients of an herb
are bound to phospholipids on a molecular level, in which the structure of the phospholipids
surrounds the active ingredients, anchoring the molecules through chemical bonds to the polar
head of the phospholipid. In the phytosome formation, the phospholipid and the individual
plant components form a 1:1 or a 2:1 complex, depending on the substance, thus enhancing its
absorption.

Advantages of Phytosomes over other conventional dosage forms

• Significative enhancement of the bioavailability of botanical extracts due to their
complexation with phospholipids and improved absorption in the intestinal tract;
• Permeation of the non-lipophilic botanical extract to allow better absorption from the
intestinal lumen, which is otherwise not possible;
• The formulation of Phytosomes is safe;
• They have been used to deliver liver protecting flavonoids because they can be made
easily bioavailable by phytosomes;
• This technology offers cost effective delivery of phytoconstituents
 • They can be also used for enhanced permeation of drug through skin for transdermal
and dermal delivery.
• The phytosomal system is passive, non-invasive and can is suitable for immediate
commercialization.
• The dose requirement is reduced due to improved absorption of the main constituent.
• the entrapment efficiency is high and more over-predetermined because the drug
itself forms vesicles after conjugation with lipid.
• They offer a better stability profile because chemical bonds are formed between the
phosphatidylcholine molecules and phytoconstituents.
• This technology has no large-scale drug development risk since the toxicological
profiles of the phytosomal components are well documented in the scientific
literature.
• Relatively simple to manufacture with no complicated technical investment required
to produce Phytosomes.
• They also have many applications in the pharmaceutical, veterinary, and cosmetic
fields.

Pharmaceutical Scope of Phytosome

• It enhances the absorption of lipid insoluble polar phytoconstituents through oral as
well as topical route showing better bioavailability, hence significantly greater
therapeutic benefit.
• Appreciable drug entrapment.
• As the absorption of active constituent(s) is improved, its dose requirement is also
reduced.
• Phosphatidylcholine used in preparation of Phytosomes, besides acting as a carrier
also acts as a hepatoprotective, hence giving the synergistic effect when
hepatoprotective substances are employed.
• Chemical bonds are formed between phosphatidylcholine molecule and
phytoconstituent, so the Phytosomes show better stability profile.
• Application of phytoconstituents in form of phytosome improves their percutaneous
absorption and act as functional cosmetics.

Clinical Studies
• “The superior bioavailability of silybin complexed with PC over non-complexed silybin
has been documented through pharmacokinetic studies conducted in rats and
humans. Figure 9 illustrates that, in rats, a large dose of silybin given orally as plain
silymarin remained virtually undetectable in the plasma for the six-hour experiment. In
marked contrast, when the same amount of silybin (200 mg per kg body weight) was
given as Siliphos®, a silybin-PC phytosome, it was detected in the plasma within
minutes, and by one hour its levels had peaked. Its plasma levels remained elevated
past the six-hour mark. The superior absorption of the silybin from Siliphos is reflected
in its clearance in the urine. Figure 9 illustrates the silybin from Siliphos remained
elevated at 70 hours following oral dosing, while the silybin given alone barely rose
above detectable levels until after 25 hours. Siliphos has been demonstrated to reach
the liver, its target organ. Silybin was substantially present in bile fluid two hours
following the administration of Siliphos and the liver continued to secrete silybin into
the bile during the entire study. Silybin, given as the non-complexed silymarin, was
barely detectable in the bile during the same period.15”
• “X. Yanyu et al. (2006) prepared a silybin–phospholipid complex to make oral
bioavailability of silybin increase and to study its physicochemical properties, and to
compare the pharmacokinetic characteristics and bioavailability after oral
administration of silybin–phospholipid complex and silybin-N-methylglucamine in rats.
They studied the solubility and found that higher solubility in water or n-octanol for
phospholipids complex than that of the physical mixture. Drugs and phospholipids of
phospholipids complex in vitro had not changed characteristics of themselves, but the
combination changed their characteristics greatly in-vivo, such as a remarkable
enhancement of GI tract absorption 77.”

• “Acute toxicity studies of silymarin after intravenous infusion have been carried out in
mice, rats, rabbits, and dogs. The LD50 values were 400 mg/kg in mice, 385 mg/ kg in
rats, and 140 mg/kg in rabbits and dogs though these values were dependent on
infusion rate. With slow infusion rate (over 2 to 3 h) the LD50 increased to 2 g/kg in
rats and after oral administration it was even 10 g/kg [23]. Silymarin is a non-lipophilic,
poorly water soluble (0.05 mg/mL) mixture of flavonolignans; it is transported bound
to serum albumin as the carrier protein [28]. Results from animal models applying CCl4
as well as other hepatotoxic agents, e.g., paracetamol, show a significant change in
hepatic acute phase enzymes and pro-fibrotic markers due to silymarin application
(Table 2) [67, 68].”

• “The current study determined the intravenous and oral bioavailability of silibinin
phosphatidylcholine complex (SPC) in cats. Oral SPC has a bioavailability of
approximately 7% in the cat. One pharmacological result of silymarin administration is
an increase in hepatic glutathione content. Specifically, one study found the
administration of silymarin to rats elicited a 50% increase in total glutathione content
which was maximal by the third day of treatment Complexing silibinin with
phosphatidylcholine increases its oral uptake and bioavailability. Administration of a
silibinin-phosphatidylcholine complex to rats increased plasma levels, was detected in
liver microsomes, and protected against lipid peroxidation. Silibinin-
Phosphatidylcholine Complex: Phosphatidylcholine is hygroscopic, and stability is
dependent on proper handling. Testing of the free powder under conditions of 37ºC
for 1 month showed no change in silibinin, an 8.5% decrease in phosphatidylcholine
content, and a slight increase in water from 2.5% to 3.8%. Under conditions of 25ºC
over 6 months there were no changes in silibinin, phosphatidylcholine or water
content. The “phytosome” complex of phosphatidylcholine and silibinin used in this
study is reported to improve bioavailability five-fold in human subjects. Oral
administration of SPC effectively raised plasma levels of silibinin in these study cats
Confirmation of the bioavailability of this complex coupled with the absence of side-
effects other than temporary sedation most likely due to the ethanol vehicle, suggests
that oral administration of SPC is a viable treatment option for felines with clinical
disease.”
• “Recently, sodium cholate/phospholipid-mixed micelles containing silibinin were
obtained and were found to be readily soluble; the maximum solubility being
10.0±1.1mg/mL. When silibinin–mixed micelles and silibinin-N-methylglucamine were
orally administered to dogs (90 mg, expressed as silibinin equivalents), it increased the
bioavailability of silibinin-mixed micelles versus silibinin-N-methylglucamine by up to
252.0% and led to a marked reduction in effective silibinin doses” (Tiwari & Mishra,
2015)
Indications
Silybin has a wide range of indications, as follows:
• Adjunctive treatment for liver disease, such as hepatitis, colangiohepatitis, those
caused by acute hepatotoxic reactions, such as mushroom poisoning by Amanita
phalloides, cirrhosis and hepatic lipidosis;
• Hepatoprotective agent against a variety of hepatotoxic substances, such as Amanita
phalloides (the “Death Cap Mushroom”), carbon tetrachloride, arsenic, and
acetaminophen, as well as in patients on long-term therapy with potentially
hepatotoxic drugs including lomustine;
• Enhancing liver regeneration through antioxidant effects, therefore supporting liver
function, decreasing liver enzymes, limiting hepatic injury associated with Amanita
mushrooms, galactosamine, carbon tetrachloride, acetaminophen, radiation, cold
ischemia, ethanol and aflatoxicosis, and as an adjunct in giardia treatment or during
metronidazole administration to decrease adverse effects;
• Regarding cancer, silybin is known to be beneficial in preventing certain types of
cancer, exerting antineoplastic effects, by reducing tumor initiation and promotion;
• Improvement of the efficacy and reduction of the negative effects of chemotherapy,
either alone or conjugated with other molecules, associations proven to reduce liver
enzymes;
• Improvement of diabetic control;
• Protection of the pancreas during pancreatitis or from drug injury;
• Improvement of hyperlipidemia.

Doses
Across the literature, it is possible to find a wide range of dosages for the use of silibinin in
veterinary. However, as most of them are extrapolated from other species (e.g., humans,
monkeys, and rodents) they can range from 15-250 mg/kg q24h, differing amongst
therapeutical indication. It must be stated that this amplitude of doses occurs due to the lack
of standardization of Milk Thistle-based products used in the studies. Despite that, most of the
scientific references point to a range of 20-50mg/kg q24h.
However, as every dosage mentioned above refers to silibinin administered in its natural form,
without any drug delivery system associated, there was the need for determining the dosage
of the silibinin-phosphatidylcholine complex (SPC), as it is known to be significatively lower.
Therefore, it has been established that the SPC complex dose of administration on small
animals is 3-6mg/kg PO q24h.
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