C h a p te r 4 E x tra c t io n , F ra c t io n a t io n and

P h y t o c h e m c a l P r o f il in g OF N a tu r a l P l a n t P ro d u c ts F o r R a d io p r o t e c t io n

41 Focus o f the Present StydyFew of the systematic approaches followed for selection, authentication and

processing of natural plant products (NPPs) followed by screening to develop an

activity profile are presented in this chapter. The present study was aimed at;

> Selection, collection, authentication and preparation o f numerous fractionated

extracts and or fractions o f different natural plant products.

> Identification o f the major active principles present in the natural plant

products using HPLC and LC-MS analysis.

> Screening o f the natural plant products on the basis o f their net reductant

power (antioxidant activity in aqueous phase)

4.2 Experimentation® The fractionated extracts using rhizome material of Leh and Ladakh High-

Altitude region were prepared by the methodology described earlier. The fractionated extracts using the solvent system i.e., n-hexane, chloroform,

alcohol (ethanol), 50% alcohol, and water were coded as HE, CE, AE, HA

and WE respectively.

« A semi-purified extract of rhizome of low-altitude Podophyllum hexandrum

(Himachal Pradesh) was prepared and coded as LAPE. Podophyllotoxin was

partially removed while preparation of LAPE. LAPE was further fractionated

into fractions i.e., benzene, ethyl-acetate, chloroform, chloroform:methanol

(90:10), chloroform:methanol (75:25), chloroform:methanol (50:50), methanol

fractions and were designated as lapBF, /apEAF, lapCF, /apCMF-1, lapCMF-

2, /apCMF-3 and lapMF respectively.

• Fresh berry extract of Hippophae rhamnoides was fractionated yielding two

fractions i.e., FRF (flavonoids rich fraction) and FLF (flavonoids lacking

fraction), confirmed by HPTLC analysis.

“ 70 % ethanolic extract of Rhodiola imbricata was also prepared using the

methodology described in Materials and Methods.

- 8 7 -

« HPLC analysis of low-altitude extract (LAPE) and five fractionated extracts of

high-altitude Podophyllum hexandrum (HAPH) for identification of aryl-tetralin

lignans.« HPTLC based qualitative analysis of the presence of different flavonoids was

also performed,

® HPLC based analysis of 70% ethanolic extract of Rhodiola imbricata was also

performed,B LC-MS analysis of LAPE, fractionated extracts of HAPH was done and the

percentage of each identified aryl-tetralin lignan was evaluated using co-

spiking analysis,

“ Polyphenolic content of different fractionated extracts of HAPH, the semi-

purified extract of LAPH and 70% ethanolic extract of Rhodiola imbricata

(REC-7004) using the method of Singleton and Rossi [1965],

« Antioxidant activity in aqueous phase was evaluated using the methodology

of Oyaziu, 1986.

® A correlation analysis was performed between the variation in values of

absorbance at least concentration tested and changes in the dielectric

constant of the solvent systems used for extraction/fractionation. This

comparative analysis was done using SPSS software version 10. The

dielectric constant of the dual solvent system was evaluated arbitrary using

the formula:Dielectric constant A:B (x:y%) =

X * dielectric constant of A+ y * dielectric constant of Bl 100

4.3 Results4.3.1 Analysis of Natural Plant ProductsThe standardization and quality control parameters investigated in case of herbal

material of Podophyllum hexandrum are illustrated in Table-4.1.The natural plant

products i.e., five extracts of HAPH, semi-purified extract of LAPH (LAPE) and its six

fractions, flavonoid rich and lacking fractions (~ two in number) of Hippophae

rhamnoides and extract of Rhodiola imbricata were prepared. The respective solvent

system and their corresponding dielectric constants are illustrated in Table-4.2.

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Table 4.1: Standardization and Quality Control of HAPH

Parameter Investigated % Limits1. Loss on drying at 105°C2. Total ash content3. Acid insoluble ash4. Microbial Counts Escherichia coli (CFU/gm)Total fungal content (CFU/gm)5. Pesticides residues(DDT, BHC, aldrin, dialdrin, melifox and toxaphene)6̂

5.94%4.0679%0,2938%

<10 ̂(within limits < 10̂ ) 7x10^ (within limits< 10'*)

Nilwithin limits

Table 4.2: Natural Plant Products Extracted In Various Solvent Systems

Name of Plant Species Code(s) Yield (%) Solvent System DielectricConstant®

High Altitude Podophyllum hexandrum (HAPH); (INIM/FRL-PH/Leh-2004)Fractionated Extracts of HAPHn-hexane Extract HE 0.560 n-hexane 2.020Chloroform Extract CE 2.140 Chloroform 4.600Alcoholic Extract AE 18.28 Ethanol (EtOH) 24,30Hydro-Alcoholic HA (AEE) 14.29 50% Ethanol 12,50Water WE 8.000 Water 80.00Low Altitude Podophyllum hexandrum (UPH); [RRL(J): 2004]Semi-Purified Extract LAPE Alcoholic 24,30Fractions of LAPEBenzene lapBF Benzene 2,280Ethyl-acetate lapEAF Ethyl-acetate 6,020Chlorofom-1 lapCMF-1 CHCl3:MeOH (90:10) 7,140Chloroform-2 lapCMF-2 CHCl3:IVIeOH (75:25) 11.70Chioroform-3 lapCMF-3 CHCl3:MeOH (50:50) 18.80Methanolic lapMF Methanol (MeOH) 33.00

Hlppophae rhamnoldes; (IHBT No, 5 and 6; INMAS/REC-1001 and REC-1002)Flavonoid Rich fraction FRF 50% EtOH 12.50

Flavonoid lacking fraction FLF Aqueous 80.00

Rhodlola Imbricata; (INM/FRL-PH/Leh-REC-7004)Rhodiola extract RE 00.40 70% EtOH 41,01

Adopted from http://www.clippercontrols.eom/info/dielectric_con5tants.html#C

4.3.2 HPLC/LC-IS Analysis of Fractionated extracts of HAPH

In the liquid chromatographic separation, it was found that a gradient of methanol (A)

and water (B) in the following manner: 1-60 min (A:B ; 65:35 ), and finally 60-70 min

(A:B ; 65:35) v/v at a flow rate of 0.6ml/min was the most optimal mobile phase. The

LC/MS pattern of reconstructed ion chromatogram of three samples of P.

hexandrum, collected at different times, matched except for a very slight variation in

the relative intensity of the peaks. HPLC profiles of different fractionated extracts are

represented in Fig,4.1, A number of aryltetrahydronapthalene and related lignans

were identified in the different fractions by analyzing the fragmentation patterns, as

revealed in corresponding tandem mass spectra. The major fragment ions observed

in the respective tandem mass spectra are summarized in Table-4,3.

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Fig. 4.1: HPLC Profiles of fractionated extracts of HAPH

Table 4.3: Lignans Identified using iX/MS profiling and LC/WiS/MS sub-structural Analysis in fractionatedextracts of HAPH

Sample Compound Retention Time (RT)

IMolecularweightM+NH4

Other fragments peak

Std 1 Podophyllotoxin 37.63 432.04 414.04, 396,93, 394,96, 352,97 312.93.

Std2HE

4'-Dem ethy Ipodophyliotoxin

4'-Demetliyipodophyi!otoxin glycoside4'-Demethylepipodophyllotoxin4'-DemetliylpodophyllotoxinPodophyilotoxin-glycosldePodophyllotoxin

28.91

28.1731.8331.8832.6736.46

418.04

580.16418.01418.01 594.15432.04

400 04382,92, 298,92

399,98,382.98,299,96,298,90,246382.96.298.92.245.89382.96.298.92.245.89 411,98,411,98,352.97,312, 95 396.93,394.96,352,97 312.93

CE 4'-DemethylpodophyllotoxlnPodopiiyllotoxin-giyooside

£p/-Podophyllotoxin Podophyllotoxin

31.24 32.39

34.03 36.01

418.04 594,18

432.06432.05

382.95,298.92,245,88432,05,416,04,397,01,352.00,312.86,261,66412.01,52.94,312.94397.00,312.94

AE 4'-Demethylpodophyllotoxin glycoside 4’-Demethylpodophyilotoxin Podophyllotoxin-glycoside ep/'podophylloloxin Podophyllotoxin

27.95

31.73 32.86 34.55 36,40

580,15

418.02594,09432,01433,05

448.91,399.91,382.96,339,08.299.74.298.93.286.89.245.72.231.02 382,94,298,89,245,67 541.98.411.98,396.94,352, 95,312.95.245.86. 412.06,352.96,312,91,118, 83396,97,394.94,353.04,312.C\C o >1 rr Oft

HA Podophyllotoxin glycoside (iso) £p/-podophyllotoxin Podophyllotoxin- glycoside Unidentified Podophyllotoxin

29.14 31.48 32.69 34.29 36.18

594.18431.91594.13312.95432.02

95.245.89411.95,352.96,312.93,286.92418.04,382.95,312.87,245.75432.02,411.97,397.00,352.94.312.92.245.87.230.90.213.86.197.84.136.89397.04.263.89.246.02

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4.3.3 HPLC/LC-iiS Analysis of LAPH

HPLC analysis revealed the presence of 16 compounds (Fig. 4.2). The phyto­

components were subjected to fragmentation analysis and the fragmentation pattern

recorded. To ensure that observed the [M+ 18]"' ion fragments of glycosylated

compounds were indeed the ammoniated species. LAPE was mixed with a diluent

consisting of 10 mM ammonium acetate in 0.1 % acetic acid. The resulting MS/MS

spectra showed an enhanced relative intensity of the [M+ ^8]* peaks for the

glycosylated compounds. Also, the subsequent MS/MS fragmentation patterns of the

sodiated and respective ammoniated species were the same.

m inFfg. 4.2; HPLC profile of LAPE

By studying the fragmentation pattern, as revealed in corresponding and tandem

mass spectra, 11 distinct peaks were obtained, out of which, 6 compounds were

identified viz., 4’-demethylpodophyllotoxin glycoside (RT: 8.05), podophyllotoxin

glycoside (RT: 12.42,13,81), podorhizol-p-D-glycoside (RT; 14.75),

deoxypodophyllotoxin (RT; 16.08-16.37), podophyllotoxin (RT; 17.26),

podophyllotoxin isomeric (RT; 20.40). Molecular weight of the seven components,

obtained online from the full ion spray mass spectrum, along with the major fragment

ions of all 11 compounds, observed in the respective tandem mass spectra, are

represented in Table-4.4.

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Table-4.4: LC-WIS Fragmentation Pattern of 11 Distinct Peaks o f LAPE

Peak Retention Time Mass Spectra CompoundNo. (min.) 1VI+ M+ NH4 and other fragments

1 8.05 562 580.2 (100), 420.0, 400.0, 383.0, 380.9 4’-demelhyl podopliylloto»n glucoside

2 12,42 576 594.2 (100), 412.0, 395.0, 312.9 Podophyllofoxin glycoside3 13.81 576 594.2 (100), 414.0, 397.0, 312.9 Podophyllotoxin glycoside4 14.75 578 596.2,594.1,418.1, 398.0 (100),

380.9, 298.9Podorhlzo(-p-D-glycoside

5 16.08-16.37 380 398 (100) Deoxypodopiiyllotoxin6 16.71-17,38 363 381 Unidentified7 17.26 414.04 432.04, 412.0, 395.0, 380.9, 312.9

(100), 298.9Podophyllotoxin

8 20,40 414.04 432.04, 414.0, 397.0, 312.9 (100), 297.9

Podophyllotoxin isomeric

9 25,57 422.04 440, 414.0, 395.0, 380.9, 312.8 Unidentified10 26.23 410 428.1, 410.9(100), 3S0.9 Unidentified11 27.95 410 428.1, 411.0 (100), 395.0, 385,0, 312,9 Unidentified

4.3.4 HPTLC Analysis o f Flavonoicl rich fraction o f Hippophae rhamnoides

A reverse phase Thin layer Chromatographic (TLC) plate of FRF fraction was

developed and compared with the standard run of kaempferol, isorhamnetin and

quercetin in parallel to it (Fig. 4.3). 3-dimensional overlay chromatogram revealed the

presence of these flavonoids as illustrated in Fig.4.4. Similar analysis with FRF

fraction exhibited no signs of these flavonoids in FLF fraction.

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KAEMPFEftOL

ISORHAMNETIN

Ci:>STANDARDS

O

SAMPLES

Fig. 4.3: Reverse phase thin layer chromatographic plate of FRF at 254nm

M l iT M d a ® JT O n m 700

Fig. 4.4: 3D overtay chromatogram of standard mix and sample track

HPLC based analysis of REC-7004 (Rhodiola imbricata)

HPLC fingerprint analysis of the hydro-alcoholic extract (50% methanol) was done at different wavelengths including 254.8nm and 340.8nm and their HPLC fingerprints are illustrated in Fig 4.5 and 4.6 respectively. The numerous peaks at A =254.8nm were found to be 3.002, 3.530, 20.065 and 23.593 min was found. At 340.8nm, peaks at time = 3.528, 20.064, 23.593, 27.544. 28.322 and 28.624 minutes, were

observed. The most common peaks include peaks at 3.5, 20.06 and 23.59 minutes respectively.

-93 -

IJ

1' 1

i1i

I ,

L M a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . . . . . . . . < . . . . . . . . . . . 1 . . . . . . . . . .

S 10 IS 20 2S

Fig. 4.5; HPLC fingerprinting of REC-7004 {Rhodiola Fig. 4.6; HPLC fingerprinting of REC-7004 (Rhodiola

Imbricata at A ~ 340.8 nm) imbricata at A = 254.8 nm)

Estimation of Polyphenolic Content

The polyphenolic content In terms of mg% of quercetin (used as standard) of

fractionated extracts of HAPH, herbal extracts of LAPH and Rhodiola imbricata

(REC-7004) and is summarized in Table-4.5.

Table 4.5; Polyphenolic Content In Different Natural Plant Products

Name of Natural Plant Product Polyphenolic Content*Fractionated Extracts of HAPHHECEAEHAWELow Altitude Podophyllum hexandrum LAPERhodiola imbricata REC-7004

0,461.259.26 2.32 1.42

1,98

0.971* Expressed In mg% of quercetin

4.3.5 Estimation of levels of aryl-tetralin lignans in P. hexandrum The levels of six compounds of aryl-tetralin lignans of LAPH are summarized in Table-4.6 while the percentages of numerous compounds of aryl-tetralin lignans of

different fractionated extracts of HAPH are summarized in Table-4.7.

Table 4.6: Estimated Content of Six Aryl-tetralin Lignans in LAPE

Estimated Qty‘'~Name of Compound4’-demethylpodophyllotoxin glycoside Podopliyllotoxin glycoside Podorhlzol-p-D-glycoslde Deoxypodophyllotoxin (RT:16.08) Deoxypodophyllotoxin (RT:16.37) Podophyllotoxin Podqptwllotoxin Isomeric

2,081,988,119.782,033,25

47.37Expressed as percentage area covered

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Table 4.7: Estimated Content of Five Aryl-tetralin Lignans in Different Fractionated Extracts of HAPH

FractionatedExtracts

Weight DIMPG" PDTG DMP“ por Podophyilotoxone

HECEAEHA

13,4460.00500.0150.5

4.9618,03

10.193,18

’ 4'-demethylpodophyllotoxin glycosider’T’odophyllotoxin glycoside;' Demethyl Podophyllotoxin; ‘‘ podophyllotoxin

11,8938,2345.3521.8

34.7 36,21 10,8711.7

38,225.250.99

4.3.6 Evaluation of Antioxidant Activity in Aqueous Phase

The reducing power of all the fractionated/semi-purified extracts of Podophyllum

hexandrum was found to increase in a dose-dependant manner as did the absorbance (Fig.4.7). The reducing power of fractions of LAPE were also compared

with LAPE and BHT (Fig.4.8). The reductant power of REC-7004 and fractions of

Hippophae rhamnoides is illustrated in Fig 4.9. The absorption unit values

(concentration to attain the unit absorbance) were calculated using the graphical values following the linear response curve maximally. Lower value is indicative of

higher reducing power. The absorption unit values of all the samples screened are summarized in Table-4.8. Butylated hydroxy toluene (BHT), a standard synthetic

antioxidant was used for comparison. The ratio of absorption unit value of BHT:

absorption unit value of natural plant product was also evaluated and enumerated in

Table-4.8.

Concontration mg/mlConcsntration mg/ml

Fig. 4.7; Reducing power of semi-purified extract LAPE and different fractions of HAPH. Absorbance at 700 nm is expressed as mean t SD. BHT, a standard synthetic antioxidant was used as control.' Significant at p<0.05 with respect to control.

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Fig 4.8 ; Reductant Power of the different fractions of LAPE; BHT was used as standard and LAPE served as reference. One-way ANOV A analysis followed by Dunnet’s t-test was used to compare t h e reductant poxwes of /apEAF as compared to other fractions.* p< 0.05 was considered as levs! of significance.

Concentration |jg/ml

Fig. 4.9 Reductant power of the fractions of Hippophae rhamnoides (FRF and FLF) and extract of Rhodiola imbrtcata (REC-7004).•Significant (pcO.OS) difference with respect to BHT.

Table 4.8: Absorption Unit Value of Natural PlantProducts Investigated in the Present Study

Code of Natural Plant Product Absorption Unit ValueHE 0.331± 0.071 0,69CE 0.075 ±0.002 3,06AE 0.038 ±0.007 6,02HA 0.044 ±0.006 5,27WE 0.351 ±0,035 0,65LAPE 0.041 ± 0,061 5,60RE 0.01 a ±0,003 12.7BHT

■ A 1 l\ / /DU-r\/ A t0.230 ±0,091 1

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4.3.7 Evaluation of Solvent Effect on Bmactivity

The correlation analysis between the change in absorbance at 1(jg/ml of the different

sets (in case of fractions of LAPE) or decrease in absorbance and the variation in

dielectric constants of the corresponding solvent systems was performed. The sets

include fractionated extracts of HAPH (Fig. 4.10); fractions of LAPE (Fig. 4.11-4.13).

The fractions of LAPE was divided into three sub-categories i.e., all fractions, fractions extracted using mono solvent system and dual solvent system. A change in

50%ethanolic fraction to aqueous fraction of Hippophae rhamnoides revealed a

perfectly negative correlation (-1). The various correlation coefficients obtained above are summarized in Table-4.9.

Fig 4.10; Correlation analysis between the fractionated extracts of HAPH and variation In the dielectric constant of the solvent system.

Fig 4.11: Variation of dielectric constant of the solvent system with respect to reductant power of the fractions of LAPE. Negative correlation (r — 0.224) was found.

0.06S o .o r?

Increase in Absorbanoo (1M0/(H|)

■aa.

i 0.020 (0I5 0.015 <.S0)□)5 0 .010 (0 .c O

0.005

7.14 <Jap C MF-1) 11,7 (Jap CMF.2) i8 .8 (lap CMF-3)

Decrease In % of Chloroform

Fig 4.12: Effect of the solvent system on the reductant power in mono-non-polar solvent system of fraction of LAPE (r = +0.975).

Fig.4.13; Effect of decrease in percentage of chloroform in dual-solvent system on the reductant power of the fractions of LAPE.

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Table 4.9: Correlation Analysis of Solvent System (Y-AxIs) and Reductant Power

"Natural Plant Products _______________ ________________________Correlation Coeffic ient ____Set-! Fractionated Extracts of HAPH +0.478Set-ll All fractions of LAPE -0.224Set-lll Fractions of 1.APE (mono non-polar system) +0.975*Set-IV Fractions of l-APE (dual solvent system) -0.545Set-V Fractions of H/ppop/iae r/7a/r?f)o/d8s ....... -1 ..... ........ ..........

'Correlation coefficient between solvent's dielectric constant and variation in reductant power (evaluated either as absorption unit value or absorbance at least concentration tested) with in the group* p<0.05 level of significance

4.4 DiscussionLiving systems maintain a state of homeostasis, which is a pre-requisite for survival.

It is well known that mammalian physiological system operates on the principles of

oxidation-reduction mechanisms. The nature of substrates/products, kinetics and

process whether it is mediated enzymatically or non-enzymatically and the

subsequent diverse interactions influence survival. Such biochemical reactions are

mostly based upon the exchange of electrons and its donation occurs at multiple

stages. Radiation-induced pro-oxidant species disrupts the equilibrium across

different compartments and induces oxidative stress. The evolutionary inherited

antioxidant defence machinery (including superoxide dismutase, catalase and

peroxidases) supports the mammalian system to tackle the various stresses

including radiation-induced free radical stress (Di Lusio, 1973; Chung et a/., 1998;

Wang et ai, 2003). However, when the intrinsic antioxidant system is overwhelmed,

exogenous supplementation of antioxidants becomes necessary to enhance the

probability of survival against radiation-induced lethality.

An in vitro system (based upon potassium ferricyanide) was used to compare the

reductant power of the samples under investigation. Fiorani and coworkers (2002)

used potassium ferricyanide to indirectly evaluate the oxido-reductase activity across

the membrane by estimation of intracellular ascorbic acid content. In the present

study, the reductant power was found to be enhanced with increase in concentrations

in all tested natural plant products. The antioxidant activity of different fractionated

extracts of HAPH, as compared to BHT, on the basis of their absorption unit values

were in the following order; AE>HA>CE>BHT>HE>WE (Fig 4,7). AE showed

maximum antioxidant potential, which could be attributed to its polyphenolic content present, which was maximally present in this fraction. On the other hand, the

fractions, viz., HE and WE exhibited lesser antioxidant potential (even lesser than that of BHT), which correlated with lower polyphenolic content of the fraction (Table

4.5). CE showed intermediate antioxidant potential, overlapping with that of BHT,

which could be attributed to its polyphenolic content (>1%) and due to the presence

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of lignans such as 4’-demethylpodophyllotoxin, ep/-podophyllotoxin, podophyllotoxin, its glycosides and other compounds (Table 4.7),

On the other hand, the antioxidant activity of LAPE was found to be nearly five times

higher than that of BHT (Table 4.8). This activity could be attributed to the presence

of lignans (aryltetrahydronapthalene) and polyphenols (1.98±0.092 mg% of

quercetin) in LAPE (Table 4.5). Lignans are known to act synergistically with a -

tocopherol leading to enhancement of vltamin-E activity (Botta et al., 2001). Vitamin-

E itself has been reported to render radioprotective effects. There Is also growing

evidence to suggest that the consumption of foods rich in lignans can indeed reduce

the incidence of several types of cancers in humans. The chemopreventive activity of

lignans is attributable to their antioxidant, and anti-estrogenic properties. In addition,

lignans prevent the production of carcinogens from oestrogens and inhibit aromatase

enzyme activity, and thereby contribute to the prevention of hormone-dependent

cancers. Since lignans exhibit a diverse array of bioactivities like anti-tumour,

hepatoprotectlve, antiviral, platelet activating factor antagonism and several other

properties they find use In the treatment of cancer and other ailments (Lampe et al.,

1994; Rios et al., 2002). Our results on the electron donation capacity correlate well

with that of reported by Wang and coworkers (2003) indicating that some constituents

present in P. hexandrum are electron donors and react with free radicals to terminate

radical chain reactions. P. hexandrum is, therefore, able to boost the natural

antioxidant defence mechanism. Similar results in case of other NPPs have been

reported by Yen and Duh (1995) and Duh and Yen (1997),

In order to understand the potentiation ability of lignans, LAPE was further

fractionated into numerous fractions and their bioactivity was analyzed. The

reductant power of /apEAF was found to be 2.48 times higher as compared to LAPE

(at 1 Mg/ml). This finding indicated that the aryl-tetralin lignans that get concentrated in

/apEAF and lapCF would be the critical factors supplementing the antioxidant

defence in overall radiation protection exhibited by LAPE. Such mechanism of the

synergistic and/or antagonistic behavior of various aryl-tetralin lignans could be

estimated by evaluating the effect of solvent system on the reductant power of the

fractions. On comparing the effect of solvent system on the reductant power, a shift

from non-polar solvent system to polar ones i.e., /apEAF to /apMF, a significant

(p<0.05) decrease in reductant power was observed (Fig 4.11). On the other hand, a

shift towards the usage of extremely non-polar solvent system (/apBF) exhibited a

significant (p<0.01) decrease in reductant power as compared to all other fractions

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(Fig 4.12). Further the usage of dual solvent system (chloroform; methanol) revealed

that a decrease In the percentage of chloroform (or increase in percentage of

methanol) exhibited a negative correlation with change in absorbance at 1|jg/ml (Fig

4.13; Table-4.9). This observation further supports the above fact. LAPE contains 4’-

demethylpodophyllotoxin glycoside (2.08%), podophyllotoxin glycoside (1.98%),

deoxypodophyllotoxin (9.78%), podophyllotoxin (3.25%), podophyllotoxin isomeric

(47.37%) (Table-4.6). The increase in the non-polarity of the solvent system

potentiates the levels of the deoxypodophyllotoxin, podophyllotoxin, and

podophyllotoxin isomeric (since Lipinski values are > 4) while an increase in the

percentage of methanol potentiates the level of glycoside derivatives of demethyl

podophyllotoxin and podophyllotoxin (Lipnski values are < 4) and the flavonoid

content. Further, such correlation analysis in case of fractionated extracts of HAPH

supports the above observation. A shift from non-polar ones to polar ones exhibited a

positive correlation (r=+0.47) i.e., an increase in reductant power (Table 4.9). But

simultaneously, the most polar fraction (WE) exhibited a significant (p <0.05)

decrease in reductant power (Fig. 4,10). These findings indicated that both flavonoids

and lignans are required in an appropriate ratio to support maximal activity.

REC-7004, 70% ethanolic extract of Rhodiola imbricata, was also found to possess

nearly twelve times higher reducing power than BHT. This lends support to the

findings of the present study that polyphenols (0.971 ± 0.01 mg%), possibly along with

other constituents (probably phenylpropanoids; a relative family of lignans i.e, rosarin

and rosavin), play a vital role in enhancing the overall electron donation capacity of REC-7004 (Table 4.8).

Evaluation of fractions of Hippophae rhamnoides revealed that the shift from aquo-

ethanolic system to aqueous system decreases the reductant power instantaneously

(Table 4.9). Thus, flavonoid enrichment is a critical factor that modulates the potency

in terms of reductant power. A significant increase in reductant power could also lead

to the generation of prooxidant activity of the natural plant products. This condition

arises if the substrate ferric ions gets converted into ferrous ions (redox active state

of iron), in case of radiation flux, the probable occurrence of such incidences is very

high. Radiation exposure destroys RBCs and thereby liberates free iron. Such

biphasic behavior needs to be addressed properly to compare the fractions at multiple stages of defence involving electronic reactions.

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LAPE, fractionated extracts of HAPH and REC-7004 exhibited a significant (p <0.05)

reduction of potassium ferricyanide to potassiunn ferrocyanide, thereby rendering

radiation protection. It also indirectly indicated their inherent ability to modulate the

oxido-reductase activity across the membranes in the mammalian system. Among

the tested NPPs, LAPE, HE, CE, AE, REC-7004 and FRF were found to be potent in

terms of reductant power and hold promise as effective antioxidant and

radioprotective agents.

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