Isolation of major Phytoconstituents and Standardization of different extracts of Boerhavia diffusa by RP-HPLC

 

Deepak Pradhan¹, Chandan Das¹, Debajyoti Das¹, Durga Madhab Kar², Goutam Ghosh*¹

¹Dept. Pharmacognosy, School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan (Deemed to be University), Kalinga Nagar, Bhubaneswar, Odisha, 751003

²Dept. Pharmacology, School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan (Deemed to be University), Kalinga Nagar, Bhubaneswar, Odisha, 751003

 

ABSTRACT:

Nature is the basketful of medicinal herbs, which consist of plethora of bio-active constituents. Natural products isolated from the plant source have been an interim part in prevention and curing of many diseases since from the era of human civilization. Numerous of natural products such as Acetylsalicyclic acid, Salicin, Morphine, Digitoxin, Quinine, Vicrestine, Vinblastine, Podophyllotoxin, and Pilocarpine have been proved to be consecrated in health care sector. Due to severe toxic effects, unavoidable side effects, and inadequate availability of medicines, World Health Organization (WHO) promotes heavily the practice of herbal medicines. So as to address this issue, a retrospective research may be required to the exploration of the bioactive constituents from the herbal sources. One such common plant is Boerhaavia diffusa belongs to family Nyctaginaceae has been explored by many research articles with a wide prospective angle to its ethnopharmacological significance, phytoconstituents, and its biological activities. The ambition of the current research was to enlighten the standardization of the different alcoholic extracts with isolated phytomarkers such as Kaempferol, Quercetin, and Boeravinone B. The standardization of the extracts was carried by reverse phase high performance liquid chromatography (RP-HPLC). The chromatograms were developed by gradient elution with 0. 05% TFA in acetonitrile as mobile phase. The samples were detected by UV detector at 340 nm. This conclusive research finds the different percentage of phytomarkers in various alcoholic extracts Boerhaavia diffusa.

 

 

INTRODUCTION:

Hippocrates II, a Greek physician of the age of Pericles (Classical Greece), who quotes “Let your food be your medicine and your medicine be your food”, which emphasize on healthy balanced diet¹. The current life style, heavily lack of such balanced diets as a consequence, the incidence of life style disorders increase by many folds^2,3. So as to resolve this issue to some extent, the western society lunch the concept of food/dietary supplements^4-8.

 

Last few decades, there is surge in the demands of standardized full spectrum extracts in the form of dietary/food supplements. A broad spectrum herbal extracts plays critical role as a potent therapeutic agent in such food/dietary supplements. Boerhaavia diffusa is one of such herbaceous plant belongs to the family Nyctaginaceae and it was named after Hermann Boerhaave in honour of respect, a famous Dutch Physician of the 18^th century^9-12. It is commonly known as ‘punarnava’ (“Punah punarnava bhawati iti” in Sanskrit translates as “that which becomes fresh again & again”) which means rejuvenating the cells or renewing the body by eliminating water soluble toxic chemicals in the form of urine and sweet as it has an advantageous role on kidney and liver¹³. It is a perennial crawling weed, prostrate or climbing herb, up to 1 m long or more and generally disseminated in the tropics and subtropics.

 

 

It is found as a common weed of the rainy season throughout India growing on roadside, waste places ascending up to an altitude of 2000m in Himalayas, distributed in wild state in Himachal Pradesh, Bihar, Bengal, Punjab, Rajasthan, Karnataka, and Tamil Nadu^{14, 15}. The plant develops lavishly in the stormy season, and organic products were getting developed in the long stretch of October to November¹⁶. The phytochemical investigation of this plant contains wide scope of secondary metabolites, for example, proteins, alkaloids, lignins, saponins, flavonoids, steroids, triterpenoids, lipids, starch, and glycoproteins^17,18. The major phytoconstituents were lunamarine, boeravinone, hypoxanthine, L-arabino-furanoside, prunol, punarnavoside, lirodendrin, palmitic acid, tetracosanoic, hexacosonoic, stearic, hentriacontane, β-ecdysone, triacontanol^19-21. The herb and roots are rich in proteins and fats. The qualitative analysis for amino acid of this herb reflects the presence of 15 amino acids, including 6 essential amino acids, while the roots restrain of 14 amino acids, including 7 essential amino acids^22,23. It has a long history of employments by indigenous and inborn individuals and in Ayurvedic or Natural home grown medication. Many of its ethnopharmacological claims cited for reproductive system, jaundice, kidney problems, skin troubles, eye diseases, wounds, and inflammation. Ayurveda characterized this plant as 'rasayana' herb which is said to have properties like enemy of maturing, restoring youth, fortifying youth, reinforcing life and intellectual competence, and sickness aversion like jaundice, all of which suggest that they increment the opposition of the body against assault¹⁸. Pharmacological studies have demonstrated that B. diffusa known to posses antifibrinolytic, antihypertensive, calcium channel antagonist hepatoprotective activities, diuretic, treatment of IUD menorrhagia, antibacterial, antiviral, antileprosy, antinematodal, anthelmenthic, antimicrobial, antispasmodic, mosquito repellent activity, cardiotonic, antistress, anti-diabetic, and anti-lymphoproliferative activities^24-27. Concern about the global demand of food supplements the current research article enlightens emphasize on the standardization of different alcoholic extracts with isolated phytomarkers.

 

MATERIAL AND METHODS:

Collection of Plant material and Preparation of extracts:

The whole herb was collected from the local market of Bhubaneswar, Odisha and was authenticated by Dr. P. K. Panda, Senior scientist, RMRC, Bhubaneswar, Odisha. The plant materials were cleaned under running tap water to remove the earthy content. The root and aerial part were separated, chopped in to small pieces and dried under shade for 7 days.

 

Figure no. 2 Schematic diagram of fractionation of methanolic extract of B. diffusa

 

The dried roots and aerial parts were powdered to coarse particles and weighed separately. The methanolic and ethyl acetate extracts of roots and aerial parts were prepared by extracting with methanol and ethyl acetate respectively. The extracts were vacuum dried to remove excess solvents and stored in desiccators. The physico-chemical properties of the different extract were evaluated in table no. 1.

 

Fractionation of alcoholic extracts:

The aqueous phase was prepared by dissolving 200gm of methanolic extract of root in the 3lt of distilled water. The aqueous phase was successive partition with different solvents with increasing the polarity as mentioned in figure no 2.

 

Isolation of phyto-constituents:

n- butanol soluble fraction was subjected to adsorption chromatography for isolation of chemical constituents. The column was eluted with chloroform and gradually gradient with methanol. A fraction 25ml was collected and monitored with TLC with mobile phase n-butanol: acetic acid: water (4:1:5) and the plate was visualize with 1% cerric ammonium sulphate. Fraction no 59-65, 83-107, and 121-149 were pulled in three separate containers and were recrystallized in methanol and acetone to give phytocomponents I, II, and III.

 

Characterization of phytoconstituents:

The isolated phytocomponents I, II, and III were characterized by various sophisticated analytical method and correlate with the literature survey.

 

Assay of different alcoholic extracts by RP-HPLC:

A waters RP-HPLC framework comprising of pumps with pump control module, with a photodiode array detector. Waters Empower programming was utilized for information investigation and information handling. The examples were investigated at 30ºC on a Merck RP-18 column (5µm, 250 x 4.0 mm ID) by UV location at 340 nm. The column was gradient elution with a binary gradient elution system with solvent A (0.05% TFA in water) and solvent B (0.05% TFA in CH₃CN) as mentioned in Table no 1. A sample strength 20 ppm concentration of the different alcoholic extracts were prepared and injected to HPLC and the chromatograms were recorded ^{28, 29}.  

 

Table no.1 Gradient elution of HPLC column

Sr. No.	Time (min)	Flow (ml/min)	%A	%B
1.	0.00	1.00	88.0	12.0
2.	25.00		79.0	21.0
3.	30.00		75.0	25.0
4.	40.00		50.0	50.0
5.	50.00		25.0	75.0
6.	60.00		10.0	90.0
7.	70.00		88.0	12.0

 

RESULT AND DISCUSSIONS:

Extraction and fractionation and isolated phytoconstituents:

The organoleptic characteristics and extractive yield for different extracts were shown in Table no. 2 and the TLC of different isolated phytocompounds are shown in Figure no 2.

 

Figure no. 2 TLC of (a) n-butanolic fraction, (b) compound I, (c) compound II, and (d) compound III

 

Table no.2 Organoleptic characteristics and percentage yield of different extracts of B. diffusa

Name of Extracts	Color	Extractive value
Boerhaavia diffusa (Roots) MeOH Ext.	Greenish	5.9
Boerhaavia diffusa (Roots) EtOAcExt	Greenish yellow	8.2
Boerhaavia diffusa (Aerial) MeOH Ext	Brown	9.5
Boerhaavia diffusa (Aerial) EtOAc Ext	Radish brown	6.8

 

Characterization of isolated phytcomponents:

Phytcomponent-I:

A yellow powder recrystalised from methanol shows the sharp melting point 276-278 ̊C. The IR spectrum shows sharp peak at 3350cm^-1 (-phenolic OH group stretching), 1674 cm^-1 carbonyl group stretching), 1489 cm^-1
(-aromatic ring stretching), 2837cm^-1 (-CH group stretching), The compound gave a fragments peaks at [M+H-H₂O]⁺=289, [M+H-H₂O-CO]⁺=257, [M+HH₂O-2CO]+ =229. [M+H-CO]⁺=275. [M+H-2CO]⁺=247. This corresponds to C₁₅H₁₀O₇. From ¹H NMR spectra (500 MHz, DMSO-d₆) δ: 6.54 (1H, d, H-8), 6.79 (1H, d H-5′), 6.80 (2H, d, H-6), 6.85 (1H, d, H-6′), 7.07 (1H, d, H-2′). ¹³C NMR (500 MHz, DMSO-d₆) δ: 97.57 (CH, C-8), 102.33 (CH, C-6), 104.65 (C, C-10), 116.67 (CH, C-2’, C-5’), 119.72 (CH, C-6’), 126.43 (C, C-1’), 132.29 (C, C-3), 146.39 (C, C-3’), 159.67 (C, C-9), 164.53 (C, C-5), 166.72 (C, C-7), 192.66 (C, C-4).

 

Phytcomponent-II:

A yellow amourphous powder recrystalised from methanol shows the sharp melting point 313-314 ̊C. The IR spectrum shows sharp peak at 3467cm^-1 (-OH group stretching), 1680 cm^-1 carbonyl group stretching), 1415cm^-1 (-aromatic ring stretching), 2838cm^-1 (-CH group stretching), ESI-MS m/z 302 [M+H]⁺, 300 [M-H]^-; ¹H-NMR (DMSO-d₆, 500 MHz) δ (ppm), 5.97 (1H, d, J = 2 Hz, H-6), 6.39 (1H, d, J = 2 Hz, H-8), 7.55 (1H, dd, J=2.0, 8.0 Hz, H-6'), 7.68 (1H, d, J = 2.0 Hz, H-2'), 9.41 (1H, s, OH-3'), 9.42 (1H, s, OH-4'), 9.53 (1H, s, OH-3), 10.72 (1H, s, OH-7), 12.39 (1H, s, OH-5), 6.81 (1H, d, J=8.5 Hz, H-5'); ¹³C-NMR (DMSO-d₆, 125 MHz) δ (ppm), 93.49 (C-8), 98.3 (C-6), 103.19 (C-10), 115.28 (C-2'), 115.82 (C-5'),120.18 (C-6'), 122.15 (C-1'), 135.91 (C-3), 145.27 (C-3'), 147.16 (C-4'), 147.87 (C-2), 156.38 (C-9), 160.98 (C-5), 164.17 (C-7), 176.19 (C-4).

 

Phytcomponent -III:

An amourphous powder recrystalised from ethanol shows the sharp melting point 207-209 ̊C. The IR spectrum shows sharp peak at 3465cm^-1 (-OH group stretching), 1663 cm^-1 carbonyl group stretching), 1412cm^-1 (-aromatic ring stretching), 2833cm^-1 (-CH group stretching), ¹HNMR: δ 5.94(CH), 6.25(CH), 6.72(CH), 6.93(CH), 7.15(CH), 9.48(OH), 10.29(OH), 10.68(OH), 11.85(OH). ¹³C NMR: δ 94.04(CH), 98.23(CH), 104.25 (C), 115.32 (CH), 117.32 (CH), 121.78(CH) 122.78(C), 136.65(C), 145.89(C), 146.5(C), 146.92(C), 158.8 (C) 161.8(C) 166.4(C), 176.1 (C).

 

Comparing the spectroscopic and chromatographic result it confirms the isolated phytcomponent I, II, III as Kaempferol, Quercetin, and Boeravinone B respectively^30-35. The percentage content of three isolated constituted molecule in different extracts are shown in Table no 3.

 

Table no.3 Quantification of different phytomarker in different extract of B. diffusa

Name of the extract	Abbreviation	Kaempferol	Quercetin	Boeravinone B
Boerhaavia diffusa (Root) MeOH	BD-R-MeOH	0.015±0.0006	0.065±0.0018	0.231±0.012
Boerhaavia diffusa (Root) EtOAc	BD-R-EtOAc	0.063±0.0009	0.015±0.0015	0.206±0.0088
Boerhaavia diffusa (Aerial) MeOH	BD-A-MeOH	0.021±0.0006	0.056±0.0009	0.233±0.01
Boerhaavia diffusa (Aerial) EtOAc	BD-A-EtOAc	0.033±0.0009	0.145±0.0048	0.247±0.0058

 

 

Figure no. 3 (a) Proton NMR (b) Mass spectra of phytcomponent –I (c) Proton NMR (d) Mass spectra of phytcomponent – II (e) Proton NMR (f) Mass spectra of phytcomponent -III

 

Figure no. 4 Percentage content of different phytomarkers in diffrent extracts Boerhaavia diffusa

 

The graphical study has revealed that peak height in terms of concentration of different phytomarkers in different extracts. The quantitative estimation of Kaempferol is found be maximum in ethylacetate extract of root and minimum in methanolic extract of the root. A significant quantity of Qucertrin and Boeravinone B are present in found in ethyl acetate extract of aerial parts and their presence are in minimum in ethylacetate extract of root.

CONCLUSION:

Mortality rate of cancer increases many folds in last few decades, which makes cancer as one of the deadliest dieses which affects to the human population. Numerous concurrent researches have been going to address this issue. The anticancer activity posses by flavonoid are back supported stack of scientific documents.  Kaempferol is a polyphenolic flavonoid is one of them, which decreases the danger of malignant growth. Epidemiological examinations delights a converse connection between kaempferol admission and occurrence of malignancy by expanding the body's cell reinforcement resistance against free radicals, which in charge of anticancer movement. Other phytomarker Boeravinone B is likewise a novel dual inhibitor of bacterial NorA efflux pump and human P-glycoprotein. Such inhibitors assume potential job to battle malignancy. The anticancer movement may additionally helped with nearness of quercetin, which has hostile to aggravation and insusceptible upgrade in vitro (cells) and in vivo (animals). So in this context current research article emphasize on dietary supplement as on the preventive measure against cancer. The ethyl acetate extract prepared from the aerial part contains higher concentration of all the three phytomarkers likes kaempferol, qucertrin, and boeravinone B. This article may provide an opportunity to develop and formulate a standardized bio-active herbal extract, with potent anticancer and immunomodulatory potentials.

 

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Received on 25.04.2019           Modified on 30.05.2019

Accepted on 27.06.2019         © RJPT All right reserved