INTRODUCTION:

Nature indeed serves as a rich storehouse of herbal remedies to cure various ailments¹. In health care programs herbal drugs are playing an essential role globally, mainly due to the universal conviction that they are without any adverse effects, moreover being economical and locally available². The realization that nature is the largest and best combinatorial library has heightened the interest in medicinal plants. Given the fact that no more than 2% of world’s reservoir of plants has been tapped, the opportunities for developing plant based drugs are seemingly limitless³.

The country has many rural parts where Western pharmaceuticals or pre-packaged Indian Ayurvedic medicines are not available. The use of traditional medicine is widespread in these rural areas, with much of the population relying on it. With over 30 tribes and as many languages, the traditional medicine culture is rich and diverse, making it an ideal site for a study of this type. The use of plant preparations in this tradition has been well documented^4-14, even though only a few sorts of plants have been evaluated for biological activity^14,15.

The Rumex species, belonging in the Polygonaceae family, include about 200 species extensively scattered around the globe. The name Rumex originated from the Latin word for dart, alluding to the shape of the leaves¹⁶. There have been numerous ethnobotanical and ethnopharmacological literature reports dealing with the occurrence and traditional uses of Rumex species^17-19.

Plant under review, Rumex hastatus is a bushy shrub belonging to the Polygonaceae family, commonly known as 'khatimal' is a perennial shrub, widely distributed in India in Himachal Pradesh, Jammu and Kashmir, Uttaranchal and Kumaun²⁰. The name Rumex L. originated from "rums" (to suck) alluding to the habit of Romans sucking the leaves to allay thirst²¹.

English : Arrowleaf Dock, Yellow Sock, Curled sock

Hindi : Churki

Kumaon: Amlora, Chulmora

Nepali : Kapu, Charimaal

Punjab : Khattimal, Katamba

Urdu : Khatti Buti

Fig 1: Rumex hastatus plant a. and b.

TAXONOMICAL CLASSIFICATION:

Kingdom : Plantae

Phylum : Magnoliophyta

Class : Magnoliopsida

Order : Polygonaceae

Family : Polygonaceae

Genus : Rumex

Species : Rumex hastatus

BOTANICAL DESCRIPTION:

Herbs are perpetual or typically annual, seldom shrubs, rarely dioeciously. Roots are typically abundant (taproots), or sometimes plants are rhizomatous. Stems vertical, climbing to horizontal, branched, not void or sulcate. Leaves are simple, habitually dimorphic, fugacious or persisting, basal and cauline, vary, periphery intact or ripple; ocrea tubular, membranous, fringe complete. Inflorescence is usually terminal, sometimes terminal and axillary, racemose or paniculate. Pedicel articulate (the useful pedicel consists of the factual pedicel and, under the joint, the pointed joint basal parts of the superficial tepals (pseudo pedicel)). Flowers are bisexual or unisexual (unisexual in dioecious, and rarely in polygamo-monoecious plants). Perianth are numerous, flattering puffy, 6 tepals and often hard-edged in fruit; valve (fruiting hub tepal) are fringe intact, erose, denticulate, or variously dentate, midvein often transformed into tubercles (tuberculate callosities). Stamens 6. Styles 3, extend; stigmas penicillate. Achenes are trigonous, elliptic to ovate^22,23.

MORPHOLOGICAL DESCRIPTION:

Shrubs are normally 50-90cm soaring. Branches are light brown in colour, lightly ridged; branchlets are green, free from hair; smooth in texture. Leaves are single or bundle of leaves growing together; petiole are 1.5-3.5cm; leaf slender triangular shape having pointed edge, 1.5-3cm × 1.5-2mm, to some extent hard, central lobe linear or closely triangular, top parts are delicately tuned, basal lobes deformed; stipules are merged into a sheath adjoining the stem fleeting, membranous. Flowers have inflorescence terminal with a branched cluster of flowers. Pedicels are inclining, articulate below central point. Flowers are polygamous. Male flowers: tepals nearly identical. Female flowers: outer tepals elliptic, reflexes in fruit; inner tepals inflated in fruit; valves pinkish, orbicular or kidney shaped, membranous, nearly pellucid, with small rounded projection at base, base deeply heart shaped, edge nearly intact, and apex imperceptive or obtuse with slight notch. Brown dry one sided seeded fruits that does not release seed with shiny, ovoid, trigonous, ca. 2mm^22,23.

TRADITIONAL USES:

Rumex hastatus is historically taken for the treatment of sexually transmitted diseases, including AIDS²⁴, laxative, tonic agent, diuretic, against rheumatism, skin diseases, piles, bleeding of the lungs, cough, headache, fever^24-32. The juice of the plant is employed for blood pressure whereas the powder of roots is beneficial for abdominal pain. Leaves have a pleasing acidic taste and utilized in chutneys and pickles³³. Anile part and contemporary tuber are utilized in tonsillitis and sore throat^14,26.

R. hastatus is employed regionally to treat jaundice and hepatitis: contemporary leaves are crushed together with water and sugar. One cup of this extract is given to the patient twice daily for 2 weeks. Leaves are directly devoured for Blood purification, Scurvy, Diuretic^23,34. Juice of leaves act as cooling, astringent, diuretic, aperients; also utilized in snakebites. Seeds are cooling, utilized in dysentery and scorpion sting. Leaves rubbed on the affected parts for relief from irritation caused by stinging nettles (Urtica dioica). Conjointly medium and consumed as vegetable^35-37. Fresh leaves are crushed and wont to stop harm from wounds^1,38. Regionally used to clean rusty vessels and as fodder for cattles. The roots and leaves of R. dentatus and R. hastatus are used for the treatment of many diseases like foot and mouth infections, asthma, cough, jaundice, headache, diarrhea, dysentery, fever, weakness and scabies^39-42.

Whole plant is acidic, slightly pungent, astringent, warm; inducing sweat and dispelling exogenous problems, moistening lung to apprehend cough; common cold, edema, asthma due to excessive phlegm. Decoction of leaves and juvenile shoots is taken thrice a day for the cure of constipation. It is commonly use as edible natural vegetables and to cure Urinary disorders, cooling, abdominal ailments like diarrhea and Constipation, worm’s problem²².

TOTAL PHENOLS, TANNINS:

The stem, bark and root of full grown R. hastatus, a herbaceous plant occurring widely in the Himalayas was evaluated for various phenol constituents. The values for total phenols, tannins and condensed tannins in the bark were about 14%, 13% and 10% respectively for two samples from different localities. About 17%, 16% and 12% for the root respectively. For the two samples of stem, the values were 8.8% and 14.8%, 7.8% and 13.6% and 6.6% and 10.2% respectively, shows the large difference. The values for tannins which bind polyvinylpyrrolidone were lower by 7-33% than values obtained with hide powder. In young plant, the levels of total phenols, tannins or condensed tannins were significantly lower and higher for the mature plant than the dead fallen stem. For industrial applications the plant appears to have a potential as a source of tannins⁵⁴.

ANTIOXIDANT POTENTIAL:

R. hastatus roots were evaluated for their antioxidant activity and phenolic content in vitro of various solvent extracts. High Performance liquid Chromatography (HPLC) was also measured. All extracts shows concentration dependent response. Butanol and methanol solvent extracts exhibited highest free radical scavenging activity in all the assays except hydrogen peroxide radical scavenging assay where chloroform fraction showed the highest scavenging ability. On the other hand, aqueous fraction showed the highest inhibition of b carotene linoleic acid peroxidation. Ethyl acetate and n-hexane extracts shows low antioxidant activity. HPLC shows the presence of glycoside, phenolic and other constituents like rutin, luteolin-7-glucoside, vitexin and luteolin. Results shows that R. hastatus roots can be use in different diseases due to presence of phenolic contents like rutin and luteolin and a good source of antioxidant²⁴.

ELEMENTAL ANALYSIS:

The ash content in R. hastatus D. Don was highest in leaves and lowest in roots. The moisture content was highest in stem and lowest in roots. Elemental analysis showed that C and O were present in all parts of R. hastatus while rest of the elements varied in different parts of plant. All Na, Mg, Al, Si, P, Cl, Ca and Fe shows their presence in root, stem and leaf of R. hastatus ⁵⁵(Hameed I. et al. 2008). R. hastatus is the best accumulator for Fe and Cr; K, Mg, Mn, Na, Cu and Pb; Ni and Cd; and Ca and Zn, respectively⁵⁶.

NUTRITIONAL ANALYSIS:

The whole plant parts of R. hastatus were estimated for its nutritional values like fat, protein, crude fibers and carbohydrates. The fat was highest in leaves (5.84%) and lowest in roots (2.54%). The protein was highest in leaves (14.00%) and lowest in stem (6.05%) Crude fiber shows highest in roots was 19.37% and lowest in leaves 14.57% Carbohydrate was maximum in fruits contains 39.24% and lowest in stems and leaves contained 24.50% carbohydrate⁵⁵.

PHARMACOLOGICAL ACTIVITY:

HEPATOPROTECTIVE ACTIVITY:

Effect of methanolic extract of R. hastatus roots (MRR) and its derived fractions, n-hexane (HRR), ethyl acetate (ERR), chloroform (CRR), butanol (BRR), and aqueous extract (ARR), was studied against carbon tetrachloride (CCl₄) induced hepato and testicular toxicity in rats. Intraperitoneal dose of 20 percent CCl₄ (0.5 ml/kg bw) was administered twice a week for eight weeks to a group of rats. Other groups were given CCl₄ and various fractions of R. hastatus roots (200mg/kg bw). CCl₄ treatment depleted glutathione contents and activities of antioxidant enzymes while increased the concentration of lipid peroxides (TBARS) along with corresponding DNA injuries and histopathological damages. Supplementation with various fractions of R. hastatus roots (200mg/kg body weight) attenuated the toxicity of CCl₄ in liver and testis tissues through improvement in the serological, enzymatic, and histological parameters towards the normal. Post treatment of R. hastatus roots (200mg/kg body weight) also reversed the alteration in reproductive hormonal secretions and DNA damages in CCl₄ treated rats. The results clearly demonstrated that R. hastatus treatment augments the antioxidants defense mechanism and provides the evidence that it may have a therapeutic role in free radical mediated diseases⁵⁷.

Furthermore investigation also revealed that R. hastatus leaves show hepatoprotective against CCl₄ induced hepatotoxicity in rats. Administration of CCl₄ in rats caused significant increase in liver function and lipid profile indicating hepatic damages which were restored by co-administration of R. hastatus extracts. Cellular and DNA damages in hepatic tissues were caused by CCl₄ which shown clear hepatic fibrosis in addition to disturb antioxidant enzyme level. Co-treatment with various fractions of R. hastatus leaves regulated these markers of oxidative dysfunctions. From the present report it was inferred that R. hastatus leaves have the ability to reverse CCl₄ – induced hepatic damages⁵⁸.

ANTIBACTERIAL, PHYTOTOXIC AND CYTOTOXIC:

The crude saponins, crude methanolic extract and subsequent fractions of R. hastatus have been evaluated for the cytotoxic potential against brine shrimps and phytotoxic potential against radish. In addition, the antibacterial activity has been evaluated for crude flavonoids (Rh.Fl), saponins (Rh.Sp), crude methanolic extract (Rh.Cr) and resultant fractions of R. hastatus by well diffusion method. In antibacterial assay, the Rh.Fl displayed considerably better activity against all the strains followed by Rh.Sp, which was comparable with positive control. Among the fractions the ethyl acetate (Rh.EaF) and chloroform fractions (Rh.CfF) revealed good zones of inhibition (ZOI). All the test samples were active against Proteus mirabilis, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa. The Rh.Cr and Rh.WtF showed moderate phytotoxicity. Based on the results obtained from the present study it may be inferred that R. hastatus may be an excellent source of natural compounds having antibacterial, anticancer and herbicidal potential especially the flavonoids, saponins, ethyl acetate and chloroform fractions⁵⁹.

Further, in vitro antimicrobial activity of R. hastatus was evaluated by the standard Disc diffusion method. All of its fractions showed antibacterial activity against Gram positive and Gram negative bacteria. Among the different fractions tested, ethyl acetate fraction was the most potent showing inhibition zones of 15mm and 7.6 mm against Staphylococcus aureus and E.coli respectively⁶⁰.

ANTINOCICEPTIVE, ANTIINFLAMMATORY AND ANTIPYRETIC ACTIVITIES:

The antinociceptive, antiinflammatory and antipyretic activities of R. hastatus D. Don stem and roots were evaluated using several experimental models. The antinociceptive activity of the ethanol and aqueous extract of stem and root was determined by using acetic acid induced writhing method, tail flick model and formalin-induced pain model in mice, using standard drugs. The antiinflammatory activity was evaluated by using carrageenan induced rat paw oedema and cotton pellet induced granuloma method and the antipyretic activity was evaluated by using the technique of yeast-induced pyrexia in Wistar rats. The ethanol and aqueous extract of stem and root at the doses of 200 and 400 mg/kg showed a significant (P < 0.001) inhibition of acetic acid induced abdominal constrictions in mice. In the tail flick model, the ethanol extract (400mg/kg) showed a significant (P < 0.001) increase in the pain threshold to the heat stimulus. The ethanol extract (400 mg/kg) of both root and stem inhibited both phases of the formalin-induced pain with a more pronounced effect on the second than the first phase. In carrageenan induced rat paw oedema method, the ethanol extracts of both stem and root (400mg/kg) exhibited significant anti-inflammatory activity. In cotton pellet induced granuloma method, the maximum percent inhibition was exhibited by ethanol extract (400mg/kg) of the stem and root, which was 23.27% and 27.25% respectively. The ethanol extracts of both the parts at the doses of 400 mg/kg, produced a pronounced antipyretic effect in hyperthermic rats in a dose dependent manner when compared with untreated rats. Moreover the ethanol extracts were more active than the aqueous extracts of both the parts in terms of activities shown in this paper. The phytochemical analysis of the ethanolic extracts of both the parts showed the presence of anthraquinone glycosides, tannins, carbohydrates, saponins and steroids. The above results have furnished the pharmacological evidence that supports the folklore claim of the drug as an analgesic, anti-inflammatory and antipyretic drug⁶¹.

NEURODEGENERATIVE DISORDERS:

The essential oil of R. hastatus was analyzed by GC-MS for the first time. The essential oil was evaluated for anticholinesterase and antioxidant assays. The anticholinesterase assay was conducted at various concentrations (62.5 to 1000μg/ml) against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Similarly, the antioxidant potential was determined using DPPH and ABTS free radicals. The GC-MS analysis of essential oil showed 123 components. The result recorded for the anticholinesterase assays demonstrated a marked potential against AChE and BChE with IC50 values of 32.54 and 97.38μg/ml respectively which were comparable with the positive control i.e., galanthamine (AChE, IC50 = 4.73μg/ml and BChE, IC50 = 11.09μg/ ml). The antioxidant assays against DPPH and ABTS free radicals also exhibited significant scavenging potential with IC50 values of 3.71 and 6.29μg/ml respectively, while for ascorbic acid the IC50 value was <0.1μg/ml against both free radicals. Based on the current investigational studies, it may be concluded that R. hastatus is an effective source of essential oil's components having anticholinesterase and antioxidant potentials, which after subjecting to drug development may lead to novel drug candidates against neurodegenerative disorders⁶²

ANTITUMOR AND ANTI‑ANGIOGENIC POTENTIALS:

The R. hastatus has been evaluated for anticancer potential against HepG2, MCF7 or LNCaP cell lines with considerable cytotoxicity. We also reported the anti-tumor and anti-angiogenic potentials of R. hastatus. The current study has been arranged to evaluate cytotoxic potential of this plant against HeLa and NIH/3T3 cell lines and sort out the most active fraction of R. hastatus along with the identification of bioactive compounds responsible for cytotoxicity. The cytotoxic potential of methanolic extract and sub-fractions of R. hastatus was performed following (3-[4,5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide) MTT calorimetric assay. Four concentrations (500, 250, 125 and 62.5μg/ml) of each sample were used against both cell lines. Two cell lines i.e. HeLa and NIH/3T3 were used in the assay. Furthermore, chemical characterization of chloroform fraction was performed by GC-MS analysis. The current investigational study demonstrates that all the solvent fractions of R. hastatus were active against HeLa and NIH/3T3 cell lines. Among all the fractions, chloroform fraction was dominant in activity against both cell lines. The observed IC50 values of chloroform fraction were 151.52 and 53.37μg/ml against HeLa and NIH/3T3 respectively. The GC-MS analysis of chloroform fraction revealed the identification of 78 compounds with the identification of bioactive ones like ar-tumerone, phytol, dihydrojasmone, sitostenone etc. It can be concluded from results that R. hastatus D. Don possess strong cytotoxic potential. Moreover, the observed IC50 values and GC-MS analysis of chloroform fraction reveal that most of the bioactive compounds are in chloroform fraction. It can be further deduce that the chloroform fraction is a suitable target for the isolation of compounds having potential role in cancer therapy⁶³.

Further investigation revealed anti-tumor and anti-angiogenic activities using potato-disc model and chorioallantoic membrane (CAM) assay respectively. Moreover, R. hastatus was also assessed for antibacterial activity against Agrobacterium tumefaciens (tumor causing bacterial strain). The positive controls used in anti-tumor, anti-angiogenic and antibacterial activities were vincristine sulphate, dexamethasone and cefotaxime respectively. The crude saponins (Rh.Sp), methanolic extract (Rh.Cr) and other solvent extracts like n-hexane (Rh.Hex), chloroform (Rh.Chf), ethylacetate (Rh.EtAc) and aqueous fraction (Rh.Aq) exhibited notable anti-tumor and anti-angiogenic activities. In potato tumor assay, the chloroform and saponin fractions were observed to be the most effective showing 86.7 and 93.3 % tumor inhibition at 1000 μg/ml with IC50 values 31.6 and 18.1μg/ml respectively. Similarly, these two samples i.e., chloroform and saponins also excelled among the entire test samples in anti-angiogenic evaluation exhibiting 81.6 % (IC50 = 17.9μg/ml) and 78.9 % (IC50 = 64.9μg/ml) at 1000 μg/ml respectively. It can be concluded that Rh.Chf and Rh.Sp might be potential targets in the isolation of natural product having anti-neoplastic action⁶⁴.

ANTIDIARRHOEAL ACTIVITY:

R. hastatus is fairly common small shrub, growing on dry slopes, rocks and walls between 700-2500m, typically in North Indian hill station. Roots of R. hastatus have been used in Ayurveda and other folk medicines for the treatment of diarrhoea. To justify its folklore, present study was undertaken to investigate the antidiarrhoeal activity of the ethanolic extract from the roots of R. hastatus (EERH). Preliminary phytochemical screening, acute toxicity study and antidiarrhoeal activity of were studied on castor oil induced diarrhoea and normal gastrointestinal models of rats at 100, 150 and 200mg/kg body weight. The preliminary phytochemical screening of the ethanolic extract from the roots of R. hastatus (EERH) results with the presence of anthraquinone glycoside, tannins, flavonoids etc., LD50> 2000mg/kg. The doses of EERH significantly decreased (P<0.001) the total number of diarrhoeal faeces. PI decreases doses dependentally (100, 150 and 200mg/kg orally) of EERH and was comparable with standard drug atropine sulphate. Conclusively, EERH has the antidiarrhoeal activity in experimental rats⁶⁵.

CONCLUSION:

R. hastatus plant has been explored exhaustively for their phytochemical and pharmacological activities. From the foregoing accounts, it is evident that R. hastatus plant has been used ethno-medicinally as a valuable therapeutic agent for a variety of diseases, as we illustrated in this article. Moreover, numerous research works have proven its uses beyond the ethno-medicinal ones in experimental animals. Various compounds which were separated from this plant may be responsible for its pharmacological activities.

CONFLICT OF INTEREST STATEMENT:

We declare that we have no conflict of interest.

ACKNOWLEDGEMENT:

The authors are grateful to the Abhilashi University, Mandi, HP, India for providing various facilities in connection with this work. The authors are also acknowledging thanks to Dr. Ajay Gautum, Director Research and Dr. Parikshit, Dean, School of Engineering, Abhilashi University, Mandi for their support and co-operation.

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Received on 16.12.2019 Modified on 13.02.2020

Research J. Pharm. and Tech. 2020; 13(8):3969-3976.

DOI: 10.5958/0974-360X.2020.00702.7

Sr. No.	Plant Part	Traditional Uses	Reference
1.	Whole Plant	Sexually transmitted diseases, including AIDS, laxative, tonic agent, diuretic, against rheumatism, skin diseases, piles, bleeding of the lungs, cough, headache, fever, blood pressure, astringent, warm; inducing sweat and dispelling exogenous problems, moistening lung to apprehend, common cold, edema, asthma due to excessive phlegm.	24, 25, 26, 27, 33, 22
2.	Leaves	Jaundice and hepatitis, Blood purification, Scurvy, Diuretic, cooling, astringent, diuretic, aperients; also utilized in snakebites, foot and mouth infections, asthma, cough, headache, diarrhea, dysentery, fever, weakness and scabies	22, 23,3 4, 35, 36, 37
3.	Roots	Abdominal pain, foot and mouth infections, asthma, cough, jaundice, headache, diarrhea, dysentery, fever, weakness and scabies	33, 39, 40, 41, 42
4.	Seeds	Cooling, utilized in dysentery and scorpion sting	36
5.	Anile part and contemporary tuber	Tonsillitis and sore throat	32, 26

Sr. No.	Chemical Constituent	Chemical Structure	Reference
1.	1,8-dihydroxy-3-methylanthracene-9,10-dione		25,53
2.	1,3,8-trihydroxy-6-methylanthracene-9,10-dione		25,53
3.	1-hydroxy-3-methyl-8-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) anthracene-9,10-dione		25,53
4.	1,6-dihydroxy-3-methyl-8-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) anthracene-9,10-dione		25,53
5.	2-(2,6-dihydroxybenzoyl)-5-methyl-3-(((2S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzoic acid		25,53
6.	Hastatuside A		25,53
7.	Hastatuside B		25,53
8.	Nepodin		25,50,53
9.	Rumexoside		25
10.	Orientaloside		25
11.	Torachryson-8-yl -beta-D-glucopyranoside		25