Isolation and Evaluation of Anti-inflammatory activity of Epigallocatechin from Senegalia rugata along with PUFAs

 

Mounica Ponugoti1,2*, Siva Prasad Panda3, Umasankar Kulandaivelu3, GSN Koteswara Rao3, Rajasekhar Reddy Alavala3, Nallaparaju Jagadeesh Varma4

1Research Scholar, College of Pharmacy, Koneru Lakshmaiah Education Foundation, Vaddeswaram,

Guntur - 522502, Andhra Pradesh, India.

2Assistant Professor, Department of Pharmacology, Hindu College of Pharmacy, Amaravathi Road,

Guntur – 522002, Andhra Pradesh, India.

3Associate Professor, College of Pharmacy, Koneru Lakshmaiah Education Foundation, Vaddeswaram,

Guntur - 522502, Andhra Pradesh, India.

4Student M.sc (Organic Chemistry), Department of Organic Chemistry, Foods, Drugs and Water,

Andhra University, Visakhapatnam– 530003, Andhra Pradesh, India.

*Corresponding Author E-mail: mounicaponuganti@gmail.com

 

ABSTRACT:

Senegalia rugata (Lam.) Britton & Rose, Synonym: Acacia concinna (Wild.) DC., Family: Fabaceae is one of the ayurvedic medicinal plant and commonly known as shikakai. The pods of S. rugata are normally used for cleansing of hair naturally due to the presence of higher content of saponins. In this study, we have isolated six compounds consisting of epigallocatechin (monomeric proanthocyanidin) from ethanol extract of S. rugata and a mixture of methyl esters of five polyunsaturated fatty acids (PUFA): methyl oleate, glyceryl trilinoleate, methyl linoleate methyl eicosenoate and methyl vernolate from petroleum ether extract of S rugata. The structures of the six compounds were elucidated using 1HNMR, 13CNMR and IR spectral studies. Epigallocatechin has shown significant in vitro anti-inflammatory property in a dose-dependent manner using the HRBC membrane stabilization method.

 

KEYWORDS: Senegalia rugata, epigallocatechin, polyunsaturated fatty acids, anti-inflammatory property.

 

 


INTRODUCTION:

Senegalia rugata (Lam.) Britton & Rose (syn: Acacia sinuate (Lour) Merr.; Acacia concinna (Wild.) DC.)1 is one of the ayurvedic medicinal plants of the Indian subcontinent that belongs to the subfamily Mimosoideae of the family Fabaceae. It is commonly known as shikakai and soap pod as it is orthodoxically used for cleansing of hair as a natural shampoo2.

 

It is most commonly grown in tropical rainforests of Southeast Asia3. The pod extract of S. rugata has shown antidermatophytic4, antimicrobial5, antioxidant, antityrosinase activity6, vaccine adjuvant property7, chemopreventive8 and cytotoxic effect on human peripheral blood mononuclear cells and human HT-1080 fibrosarcoma cells3. The aqueous extract of leaves has exhibited antioxidant, anti-platelet, and antithrombotic activity9.

 

Various compounds have been isolated and characterized from the pods of this plant such as acacic acid lactone, machaerinic acid, and its lactone, sapogenin-B, acacidiol10, acacigenin-B11, and kinmoonosides A, B, C3. Exploring more the therapeutic aspects of S. rugata, we conducted phytochemical and biological work on it. In the present study, we have isolated and characterized six compounds consisting of epigallocatechin (monomeric proanthocyanidin) from ethanol extract of S rugata and a mixture of methyl esters of five polyunsaturated fatty acids (PUFA-5): methyl oleate, glyceryl trilinoleate, methyl linoleate, methyl eicosenoate, and methyl vernolate from petroleum ether extract using 1HNMR, 13CNMR, and IR spectral analysis. In addition to this, we have evaluated the epigallocatechin for anti-inflammatory property using  human  red blood cells as a prototype of lysosome, since the plant is used traditionally for treating inflammation related diseases12.

 

MATERIALS AND METHODS:

Plant Material:

The pods of S. rugata were collected from the local market, Guntur, Andhra Pradesh, India, in December 2019. The plant species was authenticated by Dr. P. Satyanarayana Raju, Taxonomist, Department of Botany and Microbiology, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India, and a voucher specimen was deposited in our laboratory for future reference.

 

Extraction and Isolation:

The pods (1kg) were deseeded, powdered and subjected to maceration initially with pet ether for 3 days with occasional shaking and then filtered. The filtrate was concentrated under reduced pressure to get the pet ether crude (4.8g). The marc was air-dried and macerated with ethanol for 3 more days, filtered and concentrated under reduced pressure and yielded 64g of ethanolic crude extract. The pet ether crude was adsorbed on silica gel and purified by gravity column chromatography using silica gel 100-200 mesh eluting with a gradient system Ethyl acetate–n-Hexane (0:100-20:80) in which 55 fractions (P1-P55) were collected. From them, the fractions P1-P8 were colorless and doesn’t contain any compound followed by eight light yellow colored fractions that were collected (P9-P16) containing compound NP-1 [(Rf -0.5) TLC EtOAc-n-Hexane (5:95), 28mg].

 

The fractions P17-P21 contains the compound NP-2 [(Rf -0.2) TLC EtOAc-n-Hexane (8:92), 8mg], P22-P34 contains compound NP-3 [(Rf -0.4) TLC EtOAc-n-Hexane (10:90), 31mg], P35-P45 contains the compound NP-4 [(Rf -0.3) TLC EtOAc-n-Hexane (10:90), 22mg], P46-P55 contains the compound NP-5 [(Rf -0.12) TLC EtOAc-n-Hexane (10:90), 15mg]. The ethanolic crude extract was adsorbed on silica gel and purified by gravity column chromatography using silica gel 100-200 mesh eluting with a gradient system Methanol-Chloroform (0:100-100:0) in which 30 (E1-E30) fractions were collected. From them, eleven fractions E1-E11 were found to be a mixture of compounds resembled with the pet ether crude. The remaining fractions E12-E30 contains a single compound P-1 [(Rf -0.51) TLC MeOH-CHCl3 (40:60), 36.8g].

 

Characterization of isolated Compounds:

The 1HNMR spectra were recorded on a Bruker, 400MHz and 13CNMR spectra were recorded on a Bruker, 100MHz using CDCl3 and DMSO as solvents (Laila plantex, Vijayawada). IR spectra were recorded using Bruker TENSOR-27 spectrophotometer (HCOP, Guntur). Column chromatography was performed using silica gel (100-200 mesh), TLC was carried out using pre-coated MERCK silica gel and the spots were visualized by staining with iodine vapors.

 

IN VITRO ANTI-INFLAMMATORY ACTIVITY: 

HRBC membrane stabilization method:

The anti-inflammatory activity of compound P-1 was assessed by in vitro Human Red Blood Cell membrane stabilization method13. 3ml of the blood sample was collected from a healthy volunteer who has been not on NSAIDs for 2 weeks and mixed with equal volumes sterilized Alsever solution (2% dextrose, 0.8% sodium citrate, 0.5% citric acid and 0.42% sodium chloride in distilled water). The blood was centrifuged at 3000 rpm for 20min and packed cells were separated, washed and made 10%v/v HRBC suspension with isosaline. 1ml of different concentrations (50, 100, 250, 500, 1000, 2000µg/ml) of isolated compound and diclofenac sodium was mixed with 1ml phosphate buffer (0.15M, pH 7.4), 2ml hyposaline (0.36%) (instead of hyposaline, distilled water is used for control) and 0.5ml of HRBC suspension. The mixture was incubated at 37ºC for 30min and centrifuged at 3000 rpm for 20min. The supernatant was collected and estimated spectrophotometrically at 560nm. The percentage hemolysis was calculated by using the following formula

 

% Hemolysis = (Optical density of Test sample / Optical density of Control) X 100

 

The percentage protection or HRBC membrane stabilization was calculated by using the following formula

 

% Protection = 100 – [(Optical density of Test sample / Optical density of Control) X 100]

 

RESULTS AND DISCUSSION:

Identification of isolated compounds:

Compound NP-1 of pet ether extract was obtained as a thick yellow sticky solid and its molecular formula was found to be C19H36O2. IR data of this molecule has shown the presence of olefinic double bond (C=C) at 2920.5cm-1 aliphatic –CH at 2852.96cm-1 and the carbonyl stretching(C=O) at 1735.5cm-1 in its functional group region. In the fingerprint region the peaks from 722.84-974.4cm-1 correspond to the presence of unsaturation, 1095-1244cm-1 refers to C-O stretching and the peak at 1375.07cm-1 refers to aliphatic C-H vibrations. The 1HNMR spectrum of this compound depicts the presence of olefinic protons at δ (5.34-5.36ppm). The peak at δ 2.04ppm is due to CH2 nearer to unsaturation, the methoxy protons (OCH3) of ester are observed at δ 2.058ppm.

 

The signal at δ 2.3ppm refers to the methylene protons nearer to the carbonyl of ester, the peaks from δ (0.8-0.89ppm) are due to the protons of the terminal methyl and the proton signals of all the identical methylenic units (-CH2-)n are observed from δ (1.2-1.5ppm). In the 13CNMR spectrum, the carbonyl carbon (C=O) was observed at δ (175-180ppm), the signals observed in the region δ (130-135ppm) are due to olefinic carbons (C=C), the peak at δ 75ppm corresponds to the methoxy carbon (OCH3) of ester, the peaks observed between δ(30-35ppm) are due to carbon adjacent to carbonyl of ester. The signal corresponds to terminal methyl was observed at δ 14.93ppm, the signals of methylenic units (-CH2-)n  were observed in between δ (20-30ppm). From the reported spectral data, the compound NP-1 was identified as methyl (9Z)-9-octadecenoate or methyl oleate14 (Figure-1).

 

Compound NP-2 of pet ether extract was obtained as a light yellow colored oil and its molecular formula was found to be C57H98O6. IR data of the compound showed the carbon-carbon double bond stretching at 3008.13cm-1, the peaks at 2923.2cm-1 and 2854.3 cm-1 correspond to C-H stretching of saturated carbon-carbon bonds, the peak at 1742.5 cm-1 assigned to stretching of carbonyl (C=O) functionalities. The peaks at 720.9-979.4cm-1, 1096.6-1238.4cm-1, and 1373.9 cm-1 are due to unsaturation, C-O stretching and aliphatic C-H vibrations respectively. In the 1HNMR spectrum, the signals that are observed in the region δ (5.3-5.37ppm) corresponds to vinylic protons (CH=CH). In the range of δ (4.1-4.28ppm) the signals correspond to -CH2 and -CH protons of glycerol linkage. The signals of -CH2, -CH3 and the allylic protons of the fatty acid are observed in the range of δ (0.8-2.78ppm).

 

In the 13CNMR spectrum, the signals at δ 173.18ppm were assigned to the carbonyl carbon (C=O) of the glycerol functionality, the signals at δ (127.1-131.95ppm) correspond to the vinylic carbon atoms (=C-H). The signals at δ 62.1ppm and δ 62.9ppm were assigned to the CH2 and CH carbons of glycerol linkage. The numerous signals in the range of δ (14.01-34.1ppm) correspond to CH3, CH2, and allylic carbon atoms. Hence, the above spectral data confirms the triester functionality and it was identified as 2,3-Bis[[(9Z,12Z-)-octadeca-9,12-dienoyl]oxy]propyl(9Z,12Z)-octadeca-9,12-dienoate or glyceryl trilinoleate15 (Figure-1).

 

Compound NP-3 of pet ether extract was obtained as green color solid with molecular formula C19H34O2. The IR data of the compound showed the presence of olefinic (C=C) stretching at 3008.7cm-1, the peaks at 2819.6cm-1 and 2851.99 cm-1 correspond to aliphatic (C-H) stretching. The peak at 1706.25 cm-1 corresponds to the carbonyl (C=O) functionality, whereas the peaks in the fingerprint region from 721.85-937.98 cm-1 and 1098.2-1289.3 cm-1 corresponds to unsaturation and C-O stretching respectively. The 1HNMR spectrum of the compound has shown the presence of vinylic protons (CH=CH) in the range of δ (5.3-5.4ppm). In the range of δ (2.78-2.82ppm) allylic protons were observed, δ (2.32-2.36ppm) corresponds to methoxy protons (OCH3) of the ester, the signals of the methylenic units nearer to unsaturation and carbonyl group (C=O) were observed around δ (2.01-2.09ppm) and δ (1.63-1.68ppm) respectively. The signals in the region δ (0.7-1.0ppm) corresponds to the protons of the terminal methyl (CH3) and the several peaks ranging from δ (1.3-1.59ppm) corresponds to the methylenic units (-CH2-)n.

 

The 13CNMR of the compound depicts the carbonyl functionality (C=O) at δ 180ppm, the signals ranging from δ (127-131ppm) were assigned to the vinylic carbons (C=C), the peaks in the region δ (76.66-77.3ppm) corresponds to the allylic carbon present in between the two olefins. The peaks from δ (31-34ppm) correspond to allylic carbon atoms. The signals corresponding to the terminal methyl carbon (CH3) were observed at δ 14ppm and the several peaks from δ (22.5-29ppm) are due to the methylene units (-CH2-)n. The spectral data confirms the compound as methyl(9Z,12Z)-octadeca-9,12-dienoate or methyl linoleate16 (Figure-1).

 

Figure – 1: Structures of isolated compounds from S. rugata

 

Compound NP-4 of pet ether extract was obtained as dark green color solid with molecular formula C21H40O2. The IR data of the compound depicts the olefinic stretching around 2921.28cm-1, the aliphatic stretching (C-C) was observed at 2854.2 cm-1. The peaks at 1739.3 cm-1 and 1710.8 cm-1 corresponds to the carbonyl group (C=O) of an ester in its functional group region. The peaks ranging from 722.52-969.1cm-1 corresponds to the olefinic stretching (=C-H), the peaks from 1038.5-1164.7cm-1 and 1374.7cm-1 are due to (C-O) stretching and the aliphatic (C-H) stretching respectively. Interpretation of the 1HNMR spectrum of the compound reveals the presence of vinylic (CH=CH) protons around δ (5.34-5.36ppm). The signals of the methylenic protons nearer to unsaturation and carbonyl group are observed at δ 2.04ppm and δ 2.3ppm respectively. The peaks corresponding to methoxy protons (OCH3) of the ester was observed at δ 2.058ppm. The terminal methyl protons were observed at δ (0.8-0.89ppm). The signals ranging from δ (1.2-1.5ppm) were assigned to methylene units(-CH2-)n. In the 13CNMR, the signals ranging from δ (173-178ppm) were assigned to the carbonyl carbon (C=O) of the ester. The signals ranging from δ (127-130.1ppm), δ (76-77.29ppm), and δ (34-37.45ppm) correspond to the olefinic carbons (C=C), methoxy carbon (OCH3), and allylic carbon atoms respectively. The several peaks ranging from δ (24-29ppm) were assigned to the methylene units (-CH2-)n. Based on the reported spectral evidence the compound was identified as methyl (E)-icos-2-enoate or methyl eicosenoate17 (Figure-1).

 

Compound NP-5 of pet ether extract was obtained as light green color solid with molecular formula C19H34O3. The IR data of the compound revealed the carbon-carbon double bond stretching at 2953.98 cm-1 and the peaks at 2920 cm-1 and 2855 cm-1 correspond to the aliphatic (C-C) stretching. The peaks that are observed at 1737.4 cm-1, 1545.8-1458.9 cm-1 corresponds to a carbonyl group (C=O) of an ester and aliphatic C-H vibrations respectively in its functional group region. In the fingerprint region, the peaks ranging from 638.1-970.88 cm-1 and 1037.07-1212.73 cm-1 refer to olefinic (=C-H) vibrations and (C-O) stretching respectively. From the 1HNMR spectrum, the presence of vinylic protons at δ 5.35ppm, the epoxy protons are observed at δ (3.3-3.8ppm). The allylic protons present nearer to the epoxy have appeared at δ 2.79ppm. One more allylic peak was observed at δ (2.01-2.07ppm) and the peaks ranging from δ (1.25-1.9ppm) corresponds to the protons of the methylene units (-CH2-)n and the terminal methyl (CH3). The 13CNMR of this compound shows the presence of methoxy carbon (-OCH3) at δ (76.67-77.3ppm) and methylene units (-CH2-)n in the range of δ (22.67-29.68ppm). From the spectral data the compound was identified as methyl(Z)-11-[(2S,3R)-3-pentyloxiran-2-yl]undec-9-enoate or methyl vernolate18 (Figure-1).

 

Compound P-1 of ethanolic extract was obtained as a reddish-brown sticky solid and its molecular formula was found to be C31H28O12 (of basic flavonoid ring present in condensed tannin). IR data of this molecule has shown the presence of phenolic (O-H) stretching at 3351.2cm-1, phenyl (C-H) stretching at 2931.49cm-1, presence of α, β unsaturated (C-O) stretching corresponding to 1731cm-1 and the aromatic (-C=C-) stretching at 1638 cm-1 in its functional group region. The peaks in the fingerprint region 633.5-882.7cm-1 and 1013.5-1220.09 cm-1 corresponds to aromatic (C=C) and (C=O) stretching respectively. The interpretation of the 1HNMR spectrum of the compound reveals the presence of aromatic protons at δ (6.0-6.8ppm). The signals in the region δ (2.0-5.5ppm) represents the methoxy protons and the peaks from δ (0.5-1.4ppm) depict the aliphatic protons.

 

The 13CNMR spectra give the clear outlook of this molecule, in which it resembles the conventional signals of pranthocyanidin moiety. The peaks between δ (170-180ppm) were due to the α, β unsaturated (C-O), the peaks from δ (140-150ppm) corresponds to C5, C7, C8, C31 and C41 of procyanidin. The signals of the carbon C11 are observed at δ 130ppm. The peaks between δ (110-120ppm) correspond to C21, C61, and C51 of the procyanidin entity. Signals between δ (90-110ppm) corresponds to the carbons C8, C6, C61, C21, and the C4 appeared at δ (29-49ppm). The peaks between δ (55-85ppm) represent the presence of carbohydrates. Hence, based on the above spectral data compound P-1 was identified as condensed tannin epigallocatechin (EGC) having basic pranthocyanidin ring (flavanol) in its structure so-called pine tannic acid19, 20 (Figure-1).

 

Figure-2: 1HNMR spectrum of compound – P-1

 

Figure-3: 13CNMR spectrum of compound – P-1

 

Figure-4: IR spectrum of compound – P-1

 

In Vitro Anti-inflammatory activity:

Inflammation is a protective mechanism of the immune system against a toxic stimulus triggered by tissue insult or pathogenic microbes to revive the tissue homeostasis. Infiltration of neutrophils, macrophages, eosinophils, lymphocytes, and many other cells of the immune system occurs at the site of inflammation21. The interaction between toxic stimulus and plasma membrane of neutrophils or macrophages results in the release of lysosomal enzymes into the extracellular milieu due to the destabilization of the lysosomal membrane. The released lysosomal acid hydrolase cause lysis of external pathogens. However, when the inflammation is sustained and cannot restore the physiological condition results in further damage to tissues and even causes many diseases22.

 

Figure-5: In vitro anti-inflammatory activity using HRBC membrane stabilization method.

 

Since the human RBC membrane is considered similar to the membrane of lysosome this particular method is employed for evaluation of the anti-inflammatory property. Anti-inflammatory drugs such as indomethacin, phenylbutazone, aspirin, flufenamic acid, hydrocortisone, and dexamethasone were known to stabilize the lysosomal membrane22, 23. However these drugs are known to cause GI and renal side effects, the paradigm has been shifted towards natural products. The EGC isolated from S. rugata has shown a significant in vitro anti-inflammatory property using the HRBC membrane stabilization method. The percentage protection and stabilizing ability of the ECG was found to be dose-dependent and ranged from 7.68% to 41.09% when compared to standard diclofenac sodium 22.03% to 46.50% (Table 1).

 

In a study, EGC has shown the strong anti-inflammatory property by inhibiting the NF-kB activation by NOS transcription in murine macrophages24. In another study, the EGC obtained from green tea exhibited moderate anti-inflammatory properties by inhibiting IL-1B signaling pathways and COX-1, COX-2, and MMP-2 expression in human rheumatoid arthritis synovial fibroblasts25. In addition to this, the present study demonstrates that EGC also had membrane-stabilizing property, since the viability of cells depends upon the integrity of their membranes.

 


Table 1: In vitro anti-inflammatory activity of Epigallocatechin (ECG)

Concentration (µg/ml)

Diclofenac sodium

ECG

% Hemolysis

% Protection

% Hemolysis

% Protection

50

77.96 ± 0.29

22.03 ± 0.29

92.31 ± 0.49

7.68 ± 0.49

100

74.27 ± 0.38

25.72 ± 0.38

87.21 ± 0.19

12.78 ± 0.19

250

70.98 ± 0.29

29.01 ± 0.29

83.68 ± 0.24

16.31 ± 0.24

500

68.00 ± 0.25

32.00 ± 0.25

74.43 ± 0.24

25.56 ± 0.24

1000

56.23 ± 0.19

43.76 ± 0.19

66.19 ± 0.47

33.80 ± 0.47

2000

53.49 ± 0.19

46.50 ± 0.19

58.90 ± 0.24

41.09 ± 0.24


Mean ± SEM, n=3, percentage hemolysis and protection by two groups, the statistical analysis shows significant value as p<0.005

 


CONCLUSION:

In the present work, a new compound named epigallocatechin from ethanolic extract and PUFA (methyl esters of five polyunsaturated fatty acids such as methyl oleate, glyceryl trilinoleate, methyl linoleate, methyl eicosenoate, and methyl vernolate) from Pet ether extract were isolated from the pods of Senegalia rugata for the first time. The in vitro studies proved that the anti-inflammatory potential of S rugata is due to presence of epigallocatechin which has significant membrane stabilizing property. Thus, the present study may create a greater emphasis on the novel scientific pathways for further investigation on S. rugata.

 

ACKNOWLEDGMENT:

We thanks to the authority of “KL University” for providing us all facility for successful completion of research work

 

DISCLOSURE STATEMENT:

No potential conflict of interest was reported by the authors.

 

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Received on 26.06.2020            Modified on 30.09.2020

Accepted on 25.12.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(11):5739-5744.

DOI: 10.52711/0974-360X.2021.00998