Cfiapter-III

Characterization and Tbyjcity ‘EvaCuation of<Rfiodio[d dry root JLqueous % acuum (Dried

Extract

Ch a r a c ter iza tio n and Sa fe t y Ev a l u a t io n OF R h o d io l a D r y R o o t A q u e o u s

______________ _________ V acuum D ried Ex t r a c t

H erbal products are widely used not only in Asian countries but also globally

for human well being and to prepare active pharmacological compounds

(Brekhman, 1980; Fulder, 1980). But modern medicine practitioners always

expressed a fear for such preparations on account of their safety and efficacy.

There are general and herb-specific concerns regarding medicinal plants and their

toxicity and adverse effects. Some of the expressed concerns are genuine and

required to be addressed scientifically. The climatic conditions, chemical

compositions of the soil, season of collecting the plant products and method of

extraction have a significant effect on the phj^ochemicals of the raw herbal

material (Jakstas, 2003; Subramoniam, 2001) products. These variables might

have a batch-to-batch variation which effect on the bioactive principles present in

the prepared herbal formulations (Subramoniam, 2001). Hence, it is required that a

characterized and calibrated herbal preparation is used.

In herbal preparations metal pollution can result firom direct atmospheric deposition,

geological weathering or through the contaminated river and stream sediments

(Dawson and Macklin, 1998). Efficacy, safety and quality aspects of herbal products,

especially with respect to the presence of heavy metals are of concern and subject of

ongoing debates (Itankar et al, 2001; Saper et a l, 2004). Contamination of Indian

herbal products with heavy metals such as lead, mercury, cadmium and arsenic has

been reported (Saper et al, 2004). In an Indian study 31 Ayurvedic formulations were

analyzed for their arsenic content, with the exception of one remedy, all the

formulations exceeded the maximal permissible limits (Itankar et al., 2001).

Chapter -III

49

Chapter -111--------------—«

Living organisms require trace amounts of some metals, but their excessive levels can

be detrimental to them. The buildup of toxicity in the organism may however be very

gradual, and the physical effects of poisoning from the heavy metals tends to be a

very slow process and occurs over a long period of continued exposure to the source

of the toxic metal. Their accumulation in mammals can cause metal poisoning and

serious illness such as symptoms of chi'onic toxicity, renal failure and liver damage.

Hence, it becomes mandatory that the heavy metal contents in the used herbal

preparations should be within the acceptable limits.

The herbal formulations claimed to enhance physical endurance; mental functions and

non-specific resistance of the body have been termed as ‘adaptogens’ (Brekhman,

1980). During the stressful situations supplementation of such adaptogenic substances

have been shown to increase stress tolerance (Bhargava and Singh, 1981; Grover et

al., 1995; Kumar et al, 1996, 1999, 2000, 2002). In the present investigation aqueous

and alcoholic extracts of fresh and dry root of a high altitude (4000-5,000m) plant,

Rhodiola imbricata Edgew (Syn: Sedum roseum; S. imbricata; S. rhodiola), family;

Crassulaceae, were evaluated in a dose dependent fashion for their anti-stress and

adaptogenic activity. An aqueous vacuum dried extract of Rhodiola imbricata dry root

was found to possess potent adaptogenic activity in C-H-R animal model and the

minimal optimal effective dose in rats was lOO.Omg/kg body weight (Chapter-2). The

aim of this study was to partially characterize potent adaptogenic Rhodiola imbricata

dry root aqueous vacuum dried extract, determine its heavy metal content and study

acute and sub-acute toxicity in rats, if any.

3.1 M ATERIALS AND M ETHODS

S.J.J. Experimental Animals

Inbred male Sprague-Dawley rats, 12-14 weeks old, weighing 150 ± 10 g were used

for tlie study. The animal’s maintenance and other conditions were same as described

in Chapter-2.

50

Chapter -III----------------- •

3.1.2 Plant Material & Extract Preparation

Rhodiola imbricata roots were collected, in two different batches, on the two different

dates and plants firom the same area, in the month of September from hilly regions o f

Western Himalayas, where the plant grows widely under natural conditions. The Field

Research Laboratory (now named as Defence Institute of High Altitude Research),

Leh, where the voucher specimen (voucher specimen RHF 034) of the plant material

is preserved in the herbarium, carried out the ethanobotanical identification of the

plant. Further method for cleaning, washing, drying, and processing and preparation

of vacuum dried aqueous extract from dry Rhodiola roots were same as described in

Chapter -2.

3.1.3 Characterization of the Extract

To partially characterize the Rhodiola imbricata dry root aqueous vacuum dried

extract, obtained from two different batches, their HPLC finger printing, reducing

power, total phenolic and flavonoid content were estimated.

3.1.3.1. HPLC Fingerprinting

HPLC analysis of Rhodiola imbricata dry root aqueous vacuum dried extract was

performed using Waters HPLC system (Waters Corporation, USA) equipped with

Waters 515 HPLC pump, Waters 717 plus auto sampler and Waters 2487 UV

detector. Separation was performed in a symmetry C18 250 mm X 4.7 mm ID; 5|im

column (Waters, USA) by maintaining the isocratic flow rate (l.Oml/min) of the

mobile phase methanol: acetonitrile: water (20:30:50) and peaks were detected at

225nm absorbance. The standard substances gallic acid, p-tyrosol and rosin fractions

were analyzed in the same conditions. Peaks were assigned by spiking the samples

with standard substances and comparison of the retention time and UV spectrum.

51

Chapter -III

3.1.3.2. Determination of Flavonoid in Rhodiola imbricata Dry Root Aqueous Vacuum Dried Extract

Total flavonoid content of Rhodiola root aqueous extract was determined by the

method of Zhishen et al. (1999). To various concentration of the extract (100-1000

|Lig/ml), 30[a,l of 5% sodium nitrite (NaN02) and 30)il of 10% aluminium chloride was

added. The solution was incubated at room temperature for 5min. After that 200(xl

sodium hydroxide (IN NaOH) and 240(il distilled water was added to the solution.

Quercetin was used as a reference compound. The intensity of color developed was

measured spectrophotometrically at SlOnm.

3.1.3.3. Determ in ation of Reducing Power of Rhodiola imbricata Dry Root Aqueous

Vacuum Dried Extract

The antioxidant activity of Rhodiola root aqueous extract was evaluated in terms of

reducing power using the method of Oyaizu, (1986). Different aliquots of 0.1%

aqueous extract were taken and volume made to 50)iil with distilled water. To this

200)u.l of 0.2 M phosphate buffer (pH 6.5) and 0.1% potassium ferricyanide was added

and incubated at 50°C in a hot water bath for 20 minutes. After incubation 250 p.1 of

10 % trichloroacetic acid was added to the mixture and centrifuged at 3000 x g for 10

min at room temperature. The resulting supernatant was mixed with 500|j,l o f distilled

water and 100)il of 0.1 % ferric chloride, and incubated ftirther at 37°C for 10

minutes. The intensity of color developed was measured at 700nm against a blank.

Increased absorbance is indicative of increased reducing power.

3.1.3.4.Determination of Total Phenolic Content o f Rhodiola imbricata Dry Root

Aqueous Vacuum Dried Extract

The total phenolic content was estimated in the Rhodiola root aqueous extract using

Folin-Ciocalteu reagent (FCR) based assay (Singleton and Rossi, 1965). To 10^1

aliquot (taken from a Img/ml stock solution of the extract), 90|j,l of water and 500|il

of FCR were added. The mixture was allowed to stand for 5 min at room temperature,

and then 400|.il of 7.5 % sodium carbonate solution was added. The tubes were kept

52

for 30 min at room temperature (25±1°C) and absorbance of the color developed was

recorded at 765 nm. A standard gi'aph was prepared using gallic acid (0.1 mg/ml)

solution in concentrations of 1.0-8.0 |ig/ml. Total phenols (mg/g) in the Rliodiola root

aqueous extract test samples were extrapolated fi'om standard graph and expressed as

gallic acid equivalent (GAE).

3.1.4 Analysis of Heavy Metals

All the glassware’s used for heavy metal analysis were washed and soaked in 3N HCl

for 2 h and rinsed with distilled de-ionized water before use. In brief, 0.5 g of a

Rhodiola dry root aqueous herbal extract sample was transfen'ed to a 100 ml Nessler

tube and 6 ml of HNO3 (Merck, India; max. Hg 0.005 ppm) was added and heated at

150±10°C by using a digestion unit (Model Kel plus, Kes-12, Pelican, Chennai). The

sample was digested until clear solution was obtained. The digested solution was

cooled and made to the final volume of 25 ml with de-ionized distilled water. Sample

solutions were then stored in clean polyethylene bottles for metal analysis. Reagents

blank and standards were prepared and mn simultaneously. Finally, the digested

samples were used for metal analysis using GBC atomic absorption

spectrophotometer (AAS), Model 932 AA. All the samples were analyzed three times

to estimate the concentration of Arsenic (As), Lead (Pb), Mercury (Hg), Cadmium

(Cd), Zinc (Zn), Copper (Cu) and Chromium (Cr).

The standard stock solutions of 100 ppm of Pb (II), Hg (II), Cd (II), Zn (II), Cu (II)

and Cr (III) were procured from E. Merck and As (III) standard stock solution of 1

ppm was obtained from National Physical Laboratory, Delhi. Pb (II), Cd (II), Zn (II)

Cu (II) and Cr (III) levels were detemined using Flame Atomic Absorption

Spectrophotometer while As (III) and Hg (II) levels were determined using hydride

generation technique. The percentage of relative standard deviation (%RSD) in the

concentrations of analytical replicates was ± 2%. Tlie measurements were made by

using spectral lines and optimized parameters and minimum detection limits (MDL)

of the instrament as given in the Table 3.1. The MDL was defined as the lowest

analytical signal to be distinguished quantitatively at a specified confidence level

chapter -III9------------- — --------------------------------------------------------------------------------------- --------------®

53

Chapter ~IIJ

from the background signal (Kackstaetter and Heinrichs, 1997). Analyzed heavy

metal values in various herbal exti'acts were expressed in milligrams o f metals per

kilogram extract (ppm) and compared with the available maximum allowable limits

for metals in herbal products and foodstuffs.

Table 3.1: Spectral lines and instrument conditions of GBC atomic absorption spectroph­

otometer used in emission measurements and the instrumental detection

limit for the elements measured

Instrument Metals

conditions Cd Cr Cu Pb Zn As Hg

Wavelength(iim)

228.8 357.9 324.7 217.0 213.9 193.7 253.7

Slit width(iim)

0.5 0.2 0.5 1.0 0.5 1.0 3.0

Lamp Current(mA)

3.0 6.0 3.0 5.0 5.0 8.0 3.0

Calibration range (ppm) 0.5-1.5 G.5-2.0 1.0-5.0 0.5-5.0 0.5-1.5 0.01-0.05 0.025-0.1

Detection limit

(ppm)0.02 0,02 0.02 0.02 0.02 0.0004 0.0005

Flamecomposition a a a a a a a

Atomizer b b b b b b b

Measurementmode‘s

a=Air: C2H2; 1)= Standard burner; c= Concentration least square

3.1.5 Toxicity Studies

3.1.5.1 A cute Toxicity

For -acute toxicity studies, overnight fasted 48 male rats were used. The rats were

divided into four groups with each group consisting of 12 rats. One time single dose

54

of 1, 2, 5 and lOg/kg body weight o f aqueous vacuum dried extract was administered

orally to each group o f overnight fasted rats. After extract administration the animals

were provided with food and water immediately and closely observed in their cages

for any mortality and signs o f severe toxic effects such as hypo-activity, piloerection,

anorexia, salivation, diarrhea, syncope, muscle cramping, convulsions, i f any, for 24

hours and Ilirther daily for next 14 days. In each group number o f rats that died, if

any, within the period of study were noted to calculate the LD50 value.

3.1.5.2.. Sub-acute Toxicity

This study was further sub-divided into two studies.

Study I included 36 rats to determine the sub-acute toxicity after oral administration

of Ig and 2g/kg body weight o f extract for 14 days. The study consisted o f three

groups with 12 rats in each group:

Group I: Rats were given an oral dose o f Ig/kg body weiglit (ten times o f lOO.Omg/Jcg

effective dose) in 0.5 ml volume, once a day for 14 days.

Group II; Rats were given an oral dose o f 2g/kg body weight (twenty times o f

lOO.Omg/kg effective dose) in 0.5 ml volume, once a day for 14 days.

Group III: Rats were given 0.5 ml distilled water, once a day for 14 days and served

as controls.

Body weight o f the rats was recorded every day. After 14 days o f the experiment six

rats from each group were used for biochemical analysis and another six rats o f each

group for hematology and organ weight/body weight ratios.

Study II included 24 rats to detennine sub-acute toxicity after long-term

administration (30 days) o f maximal effective dose o f Rhodiola aqueous root extract.

This study consisted o f two groups with 12 rats in each group:

Group I; Rats were given daily single maximal effective dose (lOO.Omg/kg body

weight) o f extract, orally in 0.5 ml volume, for 30 days.

Chapter -III--------------------------------------------------------------------------------------------------------------------------- -

55

Chapter -JII------------------0

Group II: Rats received 0.5 ml distilled water once daily for 30 days and served as

control.

Body weight o f the rats was recorded daily. Six rats from each group were used for

biochemical analysis. Another six rats fi'om each gi'oup were used for hematology.

Organ weight / body weight ratios were determined o f all the 12 rats in each group.

Food and water were freely available to the animals during the experiment. After 14

and 30 days o f drug treatment, animals were fasted overnight and blood samples for

biochemical analysis were obtained from orbital sinus (Riley, 1960), under mild

anesthetic ether anesthesia, using capillary tubes (with and without EDTA as per

requirement). For serum preparation, blood was collected without anti-coagulant in

dried test-tube and kept at room temperature for 30 min to separate the serum from

cellular clot. The obtained seram was then used for various assays. Blood for

hematological studies was collected in the tubes containing ethylene-diamine-tetra-

acetic acid (EDTA) as an anti-coagulant. After animals were sacrificed, vital organs

(kidney, liver, spleen, adrenal, testis, heart and lung) were carefully dissected out,

cleaned of the adhering connective tissues, blotted and accurately weighed. Ratio o f

each organ to body weight was determined.

3.1.5.3 Biochemical Evaluation

y Liver Function Tests

Serum glutamate oxaloacetate transaminase (SGOT, E C 2.6.1.1) activity

SGOT activity was determined by the method o f King (1965). The determination o f

SGOT activity is based on the transamination of aspartic acid to a-ketoglutaric acid.

One of the products o f the reaction, namely, oxaloacetate is converted to pyruvate,

which is measured calorimetrically. Tubes containing 0.5 ml o f sample were

incubated for 30 min at 37°C, preferably in a shaker water bath at low speed. The

reaction in the blank tubes was stopped by adding 2 drops o f 100 % trichloroacetic

acid (TCA) and aniline citrate solution (2 g citric acid dissolved in 2 ml o f distilled

water and 2 ml aniline). Then 0.5 ml of aspartate-a-ketoglutarate (3.61 g DL- aspartic

acid, 2 g KH2PO4 and 0.6 g o f a-ketoglutaric acid in glass distilled water, volume was

56

made up to 100 ml, pH 7.4) was added to all tubes and mixed well. The tubes were

incubated at 37°C for 30 min in a shaker water bath at low speed. The reaction was

stopped by adding 2 drops o f 100 % TCA and aniline citrate solution. All the tubes

were shaken well and left at room temperature for 20 min. Thereafter, 0.5 ml o f 2, 4-

dinitrophenyl hydrazine (DNPH) solutions was added to all the tubes and mixed. The

tubes were left at room temperature tor 5 min. and 2 ml o f toluene was added and

tubes were shaken vigorously. All the tubes were centriftiged for 5 min at 2,000 rpm.

One ml of toluene layer was pipette out and 3 ml o f alcoholic KOH (2.5 % in 95 %

alcohol) was added and mixed thoroughly. Optical density was measured at 510 nm

against a blank prepared by adding 3.0 ml o f alcoholic KOH to 1.0 ml o f toluene.

Sodium pymvate was used to prepare standards o f 5-100 p-g in 1.0 ml distilled water

and processed as per procediire described above except for the addition o f 100 %

TCA. Standard graph was drawn by plotting known concentrations of pyruvate

against the coiTesponding optical density to extrapolate concentrations in unknown

samples.

Serum glutamate pyruvate transaminase (SGPT, EC 2.6.1.2) activity

SGPT activity was determined by the method ofWroblewski and LaDue (1956). The

assay system contained 1.0 ml o f buffered substrate (53.68 g o f L-alanine in 1.0 litre

of Tris-buffer {0.1 M, pH 7.5}), 0.02 ml (0.017g/dl) o f reduced nicotinamide adenine

dinucleotide (NADH), 0.02 ml o f LDH (1.2 U/ml), 0.2 ml o f sample, 0.04 ml o f L-

oxoglutarate (0.7743 g/dl). The kinetic assay was perfonned at 340 nm for 3 min, at

45 sec interval.

Alkaline phosphatase (ALP, E C 3.1.3.1) Activity

The activity o f alkaline phosphatase in the serum samples was estimated by the

method o f Lowry et al. (1954). The assay system consisted o f 0.1ml o f buffer (0.1 M

carbonate / bicarbonate buffer, pH 10), 0.1 ml o f serum and 0.5 m l o f p-nitro-phenol

phosphate (7.7 mM). The assay system was incubated at 37°C for 10 minutes. To it

was added 0.5 ml o f TCA (7.0 %) and centriftiged for 15 min at 3,000 rpm. To the 0.6

Chapter -III# — -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------®

57

Chapter-III#—------------------------- -------------------------------------------------------------- — «

ml o f supernatant was added 3.0 ml (0.05 N) of sodium hydroxide and the absorbance

read at 400 run.

Direct bilirubin estimation

The senim bilirubin was determined as per manufacturer’s instruction using

diagnostic kits obtained from RANDOX, UK.

Fasting blood glucose

In the whole blood fasting blood glucose was estimated using Ascensia Entrust blood

glucose test strips and blood glucose meter (Bayer Diagnostics India Ltd., Baroda,

Gujrat, India).

Serum lactate dehydrogenase (LDH, E.C.Ll.1.27) activity

Serum LDH activity was estimated by the method o f Kornberg (1969). The assay

mixture consisted o f 2.8ml o f pyruvate buffer (sodium pyruvate lOmM in sodium-

phosphate buffer, 0.1 M, pH 7.0), 0.1 ml of sample and 0.1 ml o f NADH (4.2 raM) in

a total volume o f 3 ml. Enzyme activity was recorded at 340 nm. In another 0.1 ml

serum sample protein concentration was estimated (Lowry et al., 1951). Enzyme

activity was calculated using molar extinction coefficient o f 6.22 x 10" M' cm'* and

expressed as n mol NADH oxidized /min/mg protein.

Total serum protein estimation

Total serum protein was estimated by the method o f Lowry et al. (1951). In 0.25ml o f

sample, 0.25ml o f water and 2.5ml o f copper reagent (50ml o f 0.1 N NaOH, 1.0ml of

1 % copper sulphate and 1.0 ml o f 1 % sodium potassium tartrate, ratio 50: 1: 1) was

added. Afterwards tubes were incubated for 15 minutes at 37°C. Then 0.25 ml o f 1 N

FCR was added and tubes were again incubated at 37°C for 30 minutes. A standard

graph was prepared using bovine serum albumin stock solution (1 mg/ml) and varied

concentration o f 20.0 -ISO.Ofxg were used to plot the graph. Absorbance was read at

750nm. The protein concentration in the test samples were extrapolated from the

standard graph and expressed as mg/ml.

58

Chapter-III

> Kidney Function Test

Creatinine estimation

Serum creatinine was estimated by the method of Bonsnes and Taussky (1945).

Creatinine reacts with picric acid in alkaline medium to form a yellowish red

complex, intensity of which is directly proportional to the concentration of creatinine

in the sample and measured at 520 nm. The tiibes for standard contained 4.0 ml o f

distilled water, 1.0 ml of standard (creatinine standard of Img / dl, in 0.01 N

hydrochloric acid), and tubes of test samples contained 3.0 ml of distilled water, 1.0

ml of semm, 0.5 ml of (2 / 3 N sulphuric acid) and 0.5 ml of sodium tungstate (10 g /

dl). Tubes were centrifuged at 2,000 rpm for 10-15 minutes. A supernatant aliquot of

2.0 ml was taken out and to it 3.0 ml of distilled water and 1.0 ml of alkaline picrate

reagent (4 parts of picric acid reagent {0.91 gm/dl or 0.04 M} and 1 part ofNaOH

{10 g/dl}) was added. Tubes were kept at room temperature for 20 minutes and the

intensity of yellowish red complex was measured at 520 nm.

Serum electrolytes (Na , estimation

Serum electi'olytes (Na" , K" ) were estimated by using Stat Profile Phox Plus Blood-

gas Analyzer (Nova Biomedical, Waltham, MA 02454-9141, USA).

> Livids Test

Total serum cholesterol estimation

Total cholesterol levels in serum were estimated by using a CHOD-PAP Diagnostic

kits, (Cat, Number E 98118), obtained from Velon Pharmacal. Pvt. Ltd., Mumbai.

Serum triglycerides estimation

Triglycerides levels in serum were estimated by using a GPO-PAP Diagnostic kits,

(Cat. Number E 96388), obtained from Velon Pharmacal. Pvt. Ltd., Mumbai.

59

Chapter -III

3.1.6 Hematological Study

Hemoglobin and other hematological parameters viz. RBC, WBC, Hematocrit, MCV

and Platelets were detennined in control and drug treated animals using semi­

automated micro cell counter Sysmex F-820 (Tea Medical Electronics Coi*poration

Ltd. Kobe, Japan) and Auto Diluter AD 270. For hematological profile the blood

containing EDTA anticoagulant (2mg/ml) was diluted with the help of Semiautomatic

diluter AD 270. Sample for WBC count was prepared by aspirating 20 (.il of the blood

during WBC mode and mixed it with 9.94 ml diluent. Dilution obtained was 1 ;500.

Diluter was shifted to RBC mode and sample for RBC coxmt prepared from 1:500

diluted mixture by aspirating 0.1ml sample and mixing it with 9.94 ml diluent. This

gave a dilution of 1; 50000. This mixture v/as swirled gently. Tlie samples for WBC

count was lysed with 3 drops of lysing reagent (Sysmex Quicklyser) and samples

were counted by F 820 cell counter by keeping appropriate sample preparation at

appropriate ports. Cell counter was calibrated with the Sysmex Controls.

3.1.7 Histology

All the vital organs (Kidney, heart, adrenal, testes, spleen, liver, and lung) of the

animals were taken out to study histo-pathological change, if any. For this the organs

were fixed in 10 % neutral buffered formalin solution. After fixation, tissues were

routinely processed, embedded in paraffin, cut in microtome setting of 4 |.mi, mounted

on glass slides, stained witli hematoxylin and eosin (McManus and Mowry, 1965) and

examined by light microscopy.

3.1.8 Statistical Analysis

Tlie results of biochemical, hematological and organ weighVbody weight ratio were

analyzed by ANOVA with correction for multiple comparisons using Bonferroni

multiple range test, using Graph Pad Prism 2.01. The results are presented as mean ±

S.E. A value of p < 0.05 was considered as statistically significant.

60

Chapter -IIIe— —------------------------------------------------------------------------------------------•

3.2. RESULTS

3.2.1 Characterization o f the Rhodiola Root Aqueous Vacuum Dried Extract

3.2.1. l.HPLC Fingerprinting

The HPLC profile of Rhodiola root aqueous extract has been given in Fig. 3.1. HPLC

analysis confirmed the presence of gallic acid, p-tyrosol, and rosin in the studied

Rhodiola vacuum dried aqueous extract. The concentration of gallic acid, p-tyrosol

and rosin were found to be 28.35, 112.24 and 145.05 |-ig/ml respectively in Img/ml of

aqueous extract.

Fig,3.1: HPLC fingerprinting of the Rhodiola imbricata dry root aqueous vacuum dried extract at 225nm

3.2.1.2 Reducing Power

The reducing power potential of tlie Rhodiola dry root aqueous vacuum dried extract was

calculated from the graph obtained. The pattern of the gi-aph is given below (Fig. 3.2).

61

Chapter -HI

Fij». 3. 2: Reducing pciwer o f the RhodioUi inihricdtd dr\ rocM aqueous vacuum diied exliacl.

The retiucing pnucr two aqucinis \acuuni dried extracts o f Rhodiola dry root

c^htained Iroiii two dilTerent batches o f roots were found to be 2.40 ± 0 .034 mg/ml.

3 .2 .1 3 . Total Phenolic Content

'Fotal phenolic contents o f the two aqueous vacuum dried extracts o f Rhodi(4a dry

root i)btained from tv\o dilTerent batches o f roots were f(umd to be 240.0 ± 10.0 mg /

g (w'/w) of extract, in terms of gallic acid equivalent.

3.2.1.4. Total F lavonoid Content

The total flavonoitl content of the two aqueous \ acuum dried extracts of Rhodiola dry

I'oot obtainetl from iwti dilTerent hatches of roots were found to be 66.7 ± 2.45 ag

Quercctin equixalent /mg extract.

3.2.2 H eavy M etal Analysis o f the E xtract

T’hc concentrations of As, Cu and Cr found in the Rhodiola dry root aqueous vacuum

dried extract was 0.41, 5.39 and 3.2 ppm, respectively. T'he concentration of other

metals Pb, Hg, C\i and Zn v\ere found to be below the minimum detection limit

(MDL) levels (Table 3.2).

62

Chapter -III

Table 3.2: Heavy metals concentration (ppm) in Rhodiola imbricata dry root

aqueous vacuum dried extract

Extract As(ppm)

Pb(ppm)

Hg(ppm)

Cd

(ppm)Zn

(ppm)Cu

(ppm)Cr

(ppm)

Aqueous Dry Rhodiola root (Vacuum dried extract)

0.41 <MDL <MDL <MDL <MDL 5.39 3.2

Permissible limits (WHO 2005)

5.0 10.0 0.5 0.3 60.0 20.0 2.0

MDL = Miniraiiin detection limits

3.2.3. Toxicity Studies

3.2,3.1. Acute Toxicity

Results of acute toxicity studies have been given in Table 3.3. Treatment of rats with

Rhodiola root aqueous extract at oral doses of 1, 2, 5 and 10 g/kg showed no mortality

within 24 hours of treatment and even after 14 days of drug treatment.

Table 3.3: Acute toxicity (LD50) o f Rhodiola imbricata dry root aqueous vacuum

dried extract adrninistered orally to rats

Dose (g/kg body weight)

D/T Mortality latency(h)

Toxicity signs

1 0/12 - None

2 0/12 - None

5 0/12 - None

10 0/12 - None

D/T = dead/treated rats.None = no toxic symptoms during the observed period.Mortality latency == time to death (in hours) after the oral administration of the extract. Rats in each dose group (n) = 12.

63

C'IkiiUcr III

.12.J.2. Siih-Acute Toxicity

Hodv weight changes of siib-aciitt“ administration o f and 2^/kj» body weight

extract d o st for 14 days

All ihc animals. ('rall_\ aeliiiiiiistcrcd I aiitl 2y/ky l^nd\ weiyhl close l\fi 14 da\s .

coiiliiuiccl lo remain acti\c and health) ihnuigliDut Ihe pei'UKl dl siudv. I'he Healed

aiul eontiDl animals gained hiul) weiytii well up to 14 da\s m cc»mparis(Mi to one

another and eontiol animals (F-'ii:. ,v3).

All t h e v a l u e s a r c m e a n ± S . I ; n l 12 r a l s iii e a c h ^ l o u p ,

^ S i a n s i i e a l K s i j i n i r i c a n l a l |’i<( ) . ( l s . m e ( ' m | ’iaMs(i i i In L 'o i i t i i tU

Fiji.3.3: Axerage hod> weight on alternate days of' rats administered 1 g and 2g/kghod\ w eight di'se o\' RliodioUi inihricatd ix>ol ac|ueous vacuum dried extract (single dose per da\') I'or 14 davs

Hiochemital and hemat(>loj»icai param eters o f sub acute administration o f lj» and

2^/kj» l>ody vveifjht extract doses for 14 days

Table 3.4 shows the hiocheniical and henuitological |’>aiameters ol' animals orall>

treated witli Is: and 2 ”/kii hod\ weiiiht extract dose, once daih for 14 davs. 4'here was

64

no significant change in any studied biochemical and hematological parameters in the experimental animal, in comparison to control animals.

Table 3.4: Effect of sub-acute Rhodiola imbricata dry root aqueous vacuum dried

extract oral administration (Ig & 2 g/kg body weight, single dose/day for 14 days) on the biochemical & hematological parameters

Chapter -III—--------------------------------------- -—-------------------------------------------- #

Parameters Control Ig/kg bodyweight

2 g/kg body weight

Cholesterol (mg/dl) 82.90 ±3.59 87.40 ±6.60 83.30±4.10

Triglycerides (mg/dl) 54.49 ± 2.30 48.49 ± 6.03 53.20 ±1.69

Creatinine (mg/di) 0.63 ± 0.07 0.68 ± 0.04 0.63 ± 0.03

Direct Bilirubin (mg/dl) 0.38 ±0.09 0.34 ±0.10 0.33 ± 0.30

Alkaline Phosphatase (lU) 8.90 ±0.30 8.60 ± 0.45 8.29 ±0.87

SCOT (lU) 31.30±2.10 30.10±3.60 32.90±2.19

SGPT (lU) 6.59 ±0.70 6.49 ±0.89 6.89 ±1.80

LDH (nmole/mg protein) 8.27 ±0.80 8.09 ± 0.39 8.10 ±0.89

Blood Glucose (mg %) 94.00 ±5.10 89.20 ±8.20 85.20 ± 1.30

Protein (g/dl) 7.30 ±0.40 7.60 ±0.35 7.29 ±0.14

Sodium (meq/1) 153.30 ±0.50 150.78 ±2.10 152.89 ±1.89

Potassium (meq/1) 4.29 ± 0.60 4.59 ± 0.20 4.50 ±0.10

WBC (xloV/1) 7.45 ± 0.20 7.50 ±0.50 7.40 ± 0.69

RBC (xloV/I) 6.50 ±0.30 6.80 ± 0.09 6.90 ±0.10

Hemoglobin (g %) 14.20 ±0.09 14.11 ±0.20 14.28 ± 0.29

Hematocrit (%) 39.30 ±1.60 38.70 ±0.30 38.50 ±0.40

MCV (fl) 59.59 ±1.10 59.78 ±0.69 58.70 ±0.70

Platelets (xlO fi/1) 713.80 ±18.90 755.80 ±9.10 736.70 ± 34.49All the values are mean ± S.E of 6 rats in each group.

65

Body weight/organ weight changes on sub-acute administration (14 days) of Ig

and 2g/kg body weight extract doses

Table 3.5 shows the organ weight / body weiglit ratio of animals treated with 1 and 2

g/kg body weight drug dose. There was no significant change observed in the organ

weight/body weight ratios of rats, in comparison to control animals.

Table 3.5: Effects of sub-acute Rhodiola imhricata dry root aqueous vacuum dried

extract oral administration (Ig/kg and 2 g/kg body weight, single

dose/day for 14 days) on the organ weight/body weight ratio of rats

Chapter -III« - --------------— — — — -------------------------------------------------- — — ---------------------------------------------------------------------------------------------------®

Organ Control Ig/kg body weight 2g/kg body weight

Liver X 10' 31.50± 1.40 30.11 ±0.90 28.50 ±0.60

Heart x 10' 3.30 ±0.10 3.30 ±0.07 3.20 ±0.04

Kidney X 10' 3.60 ±0.09 3.49 ± 0.08 3.70 ±0.05

Spleen X 10' 1.90 ±0.07 2.00 ±0.13 1.70 ±0.07

Testis X 10' 4.90 ±0.16 4.89 ±0.33 5.10 ±0.23

Adrenal x 10' 9.30 ±0.30 8.78 ± 0.29 8.40 ±0.30

Lung X 10' 4.70 ± 0.30 5.00 ± 0.09 4.50±0.10

All the values are mean ± S.E of 6 rats in each group.

Body weight changes of long-term 30 days sub-acute administration of lOOmg/kg body weight

In sub-acute toxicity of the animals orally administered 1 OO.Omg/kg body weiglit dose

for 30 days continued to remain active and healthy tlrroughout the period of study.

The treated and control animals gained body weight well in compaiison to one

another (Fig. 3.4). The lOO.Omg/kg body weight extract dose (maximal effective

dose) treated animals gained body weight well up to 30 days in comparison to control

animals.

66

Chapicr -III

Fi«. 3.4: Average hotly weight on ahernate days of rats adininisteied elTcctive dose (1 ()().() nig/kg body weight) o( Rhodiolct Inihricatd tlry root acjueous vacLiiini dried extract (single dose per day) for 30 da \s

Biochemical and hematological parameters of long-term 30 days sub-acute

Table shi)us the results on the biochemical and hematological parameters of

animals orall}' treated v\ith 1 OO.Omg/kg maximal effective adaptogenic dose, once

daily for thirtv da\s . 'fhere was no significant change in any studied bn)chemical and

hematological parameters of expei'imental rats, in comparison to control.

67

Chapter -III• - ---------------------------------------------------------------------------------------------- •

Table 3.6: Effects of sub-acute Rhodiola imbricata dry root aqueous vacuum dried

extract oral administration (lOO.Omg/kg body weiglit, single dose/day for

30 days), on the biochemical and hematological parameters of rats

Parameters Control lOO.Omg/kg body weight

Cholesterol (mg/dl) 96.20 ± 4.30 91.56 ±4.00

Triglycerides (mg/dl) 54.10 ± 1.89 52.40 ±2.20

Creatinine (mg/dl) 0.69 ±0.02 0.65 ± 0.02

Direct Bilirubin (mg/dl) 0.33 ±0.01 0.32 ± 0.01

SCOT (lU) 8.56 ±2.09 8.30±1.10

Alkaline phosphate (lU) 8.30 ±0.46 9.10 ±0.57

LDH (nmoles/mg protein) 9.89 ± 0.40 10.30 ±0.49

Protein (g/dl) 7.00 ± 0.26 7.29 ± 0.29

Blood Glucose (mg %) 108.39 ±4.93 100.10 ±4.45

WBC (xloV/1) 6.89 ±0.31 7.40 ±0.16

RBC (xloV/1) 7.40 ±0.16 7.10±0.14

Hemoglobin (g %) 15.00 ±0.25 15.10±0.10

Hematocrit (%) 42.69 ±0.65 43.45 ± 0.78

MCV (fl) 58.00 ± 0.77 57.59 ±0.83

Platelets (xlO^/l) 730.00 ± 15.20 744.00 ±16.89

All the values are mean ± S.E o f 6 rats in each group

Body weight/organ weight changes after sub-acute administration of Rhodiola imbricata dry root aqueous vacuum dried extract, single dose/day (100.0 mg/kg body weight) for 30 days

Table 3.7 shows organ weight/body weight ratio of rats treated with 1 OO.Omg/kg body

weight drug dose for 30 days. In extract treated animals there was no change in any

organ weight/ body weight ratios in comparison to control.

68

Chapter ~JI1

Table 3.7: Effect of Rhodiola imbricata dry root aqueous vacuum dried extract oral administration (100.0 mg/kg body weight, single dose/day for 30 days), on the organ weight^ody weight ratio of rats

Organ Control lOO.Omg/kg

Liver x 10" 23.30 ±0.40 24.00 ±0.49

Heart x 10' 3.00 ±0.06 3.10±0.10

Kidney x 10'^ 2.89 ±0.05 2.89 ±0.10

Spleen x 10'^ 1.78 ±0.04 1.78 ±0.02

Testis X 10'^ 4.59 ± 0.09 4.49 ±0.10

Adrenal x 10'® 8.40 ± 0.60 8.00 ±0.40

Lung X 10'^ 5.40 ±0.10 5.20 ±0.20

All the values are mean ± S.E o f 6 rats in each group.

3.2.4. Histological Evaluation o f Tissues o f Animals Treated with Ig and 2g/kg

Body Weight fo r 14 days and 100.0 mg/kg Body Weight, Single Dose/day fo r

30 days.

The adrenals, heart, kidneys, liver, lung and spleen of rats treated with Ig and 2g/kg body weight Rhodiola root extract doses for 14 days and with lOOmg/kg body weight dose for 30 days showed a normal histological appearance.

3.3. DISCUSSION

3.3.1. Characterization o f Extract

With the recent increasing interest in herbal medicine for the prevention and treatment of various illnesses, there is an increasing concern about the safety of medicinal plants. There are general and herb-specific concerns regarding medicinal plants and their ability to produce toxicity and adverse effects. The concentration of active ingredients and other chemicals in plants varies with the maturity of the plant at the time of harvest; the time of year during harvest; geography; soil conditions; soil

69

composition and its contaminants; and weather conditions. Therefore, it is required to

characterize the herbal preparation for the presence of active constituents and also to

ensure the batch-to-hatch uniformity o f the herbal extract. So far only one species,

Rhodiola rosea has been fully characterized and evaluated for its toxicity in rats and

found to be safe in acute and sub-acute toxicity studies (Brown et al., 2002). This is

the first study on characterization and safety of a biologically active root o f Rhodiola

imbricata extract. The whole vacuum dried aqueous extract of Rhodiola dry root used

in this study was first characterized using HPLC and then looked for batch to batch

variation with reference to HPLC fingerprinting, reducing power, total phenolic

contents and total flavonoid assay. The HPLC analysis confinned the presence o f

gallic acid, p-tyrosol and rosin in the studied aqueous extract of Rhodiola root. The

HPLC fingerprinting of two extracts obtained from two different batches o f root

showed similar HPLC profiles. The variation in major peak areas of HPLC profiles

was within 2% variation. Analysis o f two batches of total flavonoid content in

Rhodiola root aqueous extract was found to be 66.7|Xg Quercetin equivalent/mg

extract, with Quercetin as standard flavonoid. This showed that there was a significant

amount of flavonoids present in the aqueous extract of Rhodiola root. The reducing

power (2.0 ± 0.04 mg/ml) of the extracts obtained firom different batches was also not

very different. The extract of Rhodiola root was also found to be rich in high phenolic

content (240.0 ± lO.Omg/g gallic acid equivalent) and batch-to-batch variation in

phenolic content was less than 5%. Phenols are one of the important phytochemical

responsible for various bioactivities and might be responsible for the observed

adaptogenic activity. It has been shown earlier that poly-phenolic compounds present

in plant preparations posses potent anti-oxidative activity (Hagerman et al., 1998).

The observed results suggested that aqueous vacuum dried extract o f Rhodiola

imbricata dry root found to posses potent adaptogenic activity and had no significant

batch-to-batch variations.

3.3.2 Heavy Metals

Heavy metals contamination in herbal products is of concern. Using herbs in medical

treatment of various illnesses one should be aware that apart from the

Chapter -III•------- — -------------------------------------------------------------------------------------------------------------------------------- ®

70

Chapter -III----------------- 9

pharaiacological effect they could turn out to be toxic because of the presence of heavy metals like Pb, Cd, Zn, Ni and other impurities. Metal pollution can result from direct atmospheric deposition, geological weathering or through the water sources due to solubility of metal ions and conditions in which the medicinal plants are grown, collected, dried, processed, stored, transported and manufacturing processes when the ready-made medicinal products are produced. In the present study the heavy metal

analysis was carried out in the Rhodiola dry root aqueous vacuum dried extract found to possess adaptogenic potential. The estimated arsenic concentrations in aqueous

extracts of diy Rhodiola root (vacuum dried) were 0.41 ppm. The WHO (2005) proposed 5ppm limit for arsenic in herbal medicines and products and the observed arsenic concentration in the herbal extracts was well below the proposed limits (WHO 2005). It suggested that the evaluated extract was free from arsenic toxicity. The estimated lead concentration in aqueous extract of dry Rhodiola root (vacuum dried) was found to be below minimum detectable limits (MDL). WHO (2005) has recommended tlie proposed limit of 1 0 ppm for lead suggesting that the extract was free of lead contamination also.

In a study in USA high level of heavy metals including mercmy were reported in Indian Ayurvedic products that put users at risk of metal poisoning (Saper et al., 2004). The evaluated extract of Rhodiola root showed the presence of mercury level below MDL and the WHO (2005) proposed limit is 0.5 ppm. The WHO (2005) proposed a 0.3 ppm limits for cadmium in herbal medicines and products. The cadmium concentration in the evaluated herbal extracts was below the MDL values. Similar observations were made by Letchamo et al. (2002) for cadmium concentrations in Seabuckthom berries. The recommended permissible maximum tolerable daily intake (PMTDI) of Zinc (Zn) is 60 mg d"’ (WHO, 1982). In the present study the estimated zinc concentrations in all the herbal extract were below the MDL values. In Indian leafy and non-leafy vegetables Copper (Cu) levels of 1.3045 and 0.5256 |ig g' have been reported (Tripathi et al., 1997). The permissible maximum tolerable daily intakes (PMTDI) of Cu recommended by the Joint Expert Committee on the Food Additives (JECFA) for a 60 kg person is 30 mg d'* (WHO, 1982a) and WHO (2005) proposed limits for Cu is 20.0 ppm, in herbal medicines and products.

71

Chapter -III------------------0

The estimated Cu concentration studied in the Rhodiola root extract found was 5.39

ppm, below the proposed limits by the WHO (2005). Results suggested that the

evaluated extract was free of mercury, cadmium, zinc and copper toxicity. The WHO

(2005) proposed limit of 2.0ppm for chromium in raw dietary herbal medicines and

products. The estimated chromium level in the aqueous vacuum dried extract of

Rhodiola imbricata dry root was found to be 3.2ppm, which was slightly more than

the WHO (2005) proposed limit of 2.0 ppm. Results suggested that Rhodiola

imbricata dry root extract was free of heavy metal toxicity and this may be on account

of the clean and pollution free high altitude environment where the Rhodiola plant

grows wildly.

3,3.3. Safety Studies

Increasing interest in herbal medicine for the treatment and prevention of vaiious

illnesses has raised the concern about the safety of medicinal plants. The first and

most important requirement of any substance, either of herbal or synthetic origin, used

for therapeutic or nutritional supplementation is to be safe and should not have any

biological toxicity. The most widely used criteria for the toxic action of a drug in

animals is decrease in body weight gain, changes in organ weights/body weight ratio

and detection of histopathological abnormalities in the vital organs. In the present

study, no such adverse effects were observed after the sub-acute dose administration

for 30 days of maximal effective lOO.Omg/kg body weight adaptogenic dose. Even

after the sub-acute administration of Ig/kg and 2g/kg body weight doses (10 times

and 20 times of the maximal effective dose) for 14 days there were no significant

changes in body weight gain, organ weight^ody weight ratio and histopathological

abnormalities of organs of the treated rats, in comparison to control animals. The

comparable body weight gain of the experimental animals with controls suggested no

gross toxicity. The comparable organ weight^ody weight ratios of all the organs of

experimental and conti'ol animals suggested that there was no gross toxic effect of the

extract on any vital organs when the maximal effective dose was administered for 30

days and also the 10 and 20 times of the maximal effective doses were administered

for 14 days. No gross histological changes were observed in all the organs studied in

72

Chapter -III•.......................... —--------- — — -------------------------------------------------------- •

both the sub-acute 30 days administration of maximal effective adaptogenic dose (lOO.Omg/kg body weight) and the sub-acute 14 days administration of Ig/kg and 2g/kg body weight doses, 10 times and 20 times of the maximal effective dose. The results indicated that the use of the herbal extract preparation in the used doses did not cause any toxicity in any of the organ studied.

In the present study, the sub-acute 30 days administration of 100.0 mg/kg body weiglit

dose, and 14 days administration of Ig/lcg and 2g/kg body weiglit doses showed no significant change in any of the studied hematological parameters and biochemical parameters related to lipid metabolism (serum triglycerides, serum cholesterol), seimii LDH, and creatinine levels in comparison to control animals. This suggested that sub-acute administration of Rhodiola imbricata dry root aqueous extract had no effect on cell membrane pemieability, muscle metabolism and kidney function.

Liver plays a central role in the detoxification of drugs. It regulates the level of most of the circulating biochemicals, drugs and toxic chemicals in association with the kidney to clear the blood of drug and toxic substances. The liver function tests viz. serum bilirabin, glutamic pyruvic transaminase (GPT), glutamic oxaloacetate transaminase (GOT), alkaline phosphatase (ALP) showed no significant change suggesting no hepatotoxicity in rats due to administration of Rhodiola root aqueous extract at higher doses of Ig/kg and 2 g/kg body weight (single dose per day, for 14 days).

Acute toxicity refers to the adverse effects occuning within a short time of 24 hours after oral administration of a single dose of relatively large amounts of test substance. The objective of acute toxicity studies was to determine the median lethal dose (LD50) after a single dose administration. LD50 is a value used to show that 50% of the animals exposed to a specific amount of a substance died consequent to the treatment.In the present study the oral LD50 of Rhodiola root aqueous extract in rats was observed to be > 10 g/kg body weight, as even after single dose treatment with 5 and 10 g/kg body weight dose no rat died in >24 h after the extract administration. This suggested low toxicity of Rhodiola imbricata dry root aqueous vacuum dried extract, as larger is the LD50 value the lower is the toxicity.

73

3.3.4. Hematological Parameters

In the present study, no significant change was observed in any hematological

pai'ameters of the animals treated with the Rhodiola root extract at a dose of Ig and

2g/kg body weight administered for 14 days and also lOO.Omg/kg body weight for 30

days. The observed findings further strengthened the earlier results that extract

administration in rats had no adverse effect.

3.4 CONCLUSION

There was no batch-to-batch variation of the aqueous extract of Rhodiola imhricata dry

root. The extract was free of heavy metal toxicity. Tlie LD50 value of the maximal

effective adaptogenic dose of vacuum dried aqueous extract of Rhodiola imhricata dry

root in rats was > lOg/kg body weight while its minimal effective observed dose was

lOO.Omg/kg body weight suggesting that the evaluated extract was safe with a large range

of therapeutic index >100. The results on long-temi administration of 10 and 20 times of

effective doses suggested that even tlie use of high doses of Rhodiola imhricata dry root

aqueous vacuum dried extract was also safe and had no toxicity in rats.

Chapter -111®— ---------------------------------------------------------------------------------------- — ---- •

74