INTRODUCTION:

Aerva lanata (L) is a tropical plant which is grown extensively in “India, Arabia, Africa, Sri Lanka, Phillipines and Java”. It is a perennial weed belonging to the family Amaranthaceae. Aerva lanata is a traditional medicine which holds to cure a various disorder such as “helminthic, diabetes, inflammation, skin diseases, kidney stones, headache, cough, cholera, dysentery and diarrhea”. Aerva lanata is extensively utilized by progressive scientific techniques which is reported to possess activities such as “diuresis¹, urolithiasis², anti-inflammatory³, antimicrobial⁴, anti-diabetic⁵, nephroprotective⁶ hepatoprotective^7,8, anthelminthis⁹ and antidiarrheal”¹⁰.

It is Proposed that “polyphenols, flavonoids, triterpenoids, and saponins[α amyrin, β amyrin, lupeol]” constituents exhibit anti-urolithiasis activity and diuretic activity^11,12. Crataeva nurvala, Crateva magna portions of root bark possessed to showed anti-urolithiasis activity¹³. Urolithiasis is a prevailing disorder with combined effect of epidemiological, genetic risk and biochemical factors by multi-factorial formation of kidney stones¹⁴. Various synthetic compounds such as butyl hydroxy anisole and butyl hydroxy toluene are broadly utilized as antioxidants in many food industries. Report of scientific analysis shows that long-term exposure or high doses to compounds can be poisonous to animals¹⁵. Polyphenolic compounds in medicinal plants is considered as a good source for natural compounds with antioxidant activity and has high potential for antioxidant compared to fruits and vegetables. Utilization of such products probably prevents formation of free radical mediated cell damage and thereby protects from several health related problems^16-18. The phytochemical constituents like Alkaloids, Flavonoids, lupeol, benzoic acid and tannic acid present in Aerva lanata are reported to have various beneficial activities¹⁹. Antidiabetic and antihyperglycemic effects are also observed from the alcoholic extracts of Aerva lanata⁵. Proteus species (urease positive microorganisms) infects urinary tract with struvite stone in which enzyme splits urea and converts to urea and carbon dioxide²⁰. Calcium oxalate reduction in albino rats is also observed in plant’s species like Phyllanthus niruri and Aerva lanata²¹.

Taxonomy ⁽²²⁾:

Domain : Eukaryote

Kingdom : Plantae

Phylum : Angiosperms

Class : Mangoliophyta

Order : Caryophyllales

Family : Amaranthaceae

Genus : Aerva

Species : lanata

Morphology:

The Perennial Shrub of Aerva lanata (Mountain Knot grass) is a shrub with woody root and soft spikes like flowers, with oval leaf of 0.5-1.5 in length arranged alternatively. The two lobes of whitish flowers with red bases are 0.1 m long alongside with pink, green and white flowers. This self-pollinating, bisexual plants is cultivated above 90-meter sea level and grown only around tropical climatic conditions²³. It has subsessile head of axillary with spike length of 6-13 mm long forming globose like structures and long membranous perianth of 1.5 m long with oblong sepals, sometimes apiculate and two stigmas [0.85 mm diameter] polished, black seed²⁴.

Phytochemical Analysis:

Screening for the stem of Aerva lanata is carried by standard protocol ^25,26. It contains phenolic compounds, flavonoids, saponins, carbohydrates, oil, phytosterols, alkaloids, tannins and proteins.

Analysis of phenolic content:

Estimation of total phenolic content:

Folin catechu reagent method²⁷ was used to determine Aerva lanata. Various concentrations of “125, 250, 500 and 1000 µg” was obtained by diluting crude extracts in methanolic solution. 50µL of extract was mixed with 2.5 ml of 7.5% Na₂Co₃ which was incubated at 45^oC for 15 mins and absorbance was measured at 765 nm. Na₂Co₃solution [ 2 ml 7.5% Na₂Co₃in 2.5 ml of distilled water] was used as blank solution. Results obtained were expressed in Gallic acid equivalence at µg concentration. Folin ciocalteu phenol method besides its range of applications has several interfering substances including “sulfur dioxide, ascorbic acid, sugar, aromatic amines, proteins, and non-phenolic organic substances” which reacts primarily with folins reagent²⁸.

Determination of phenolic compounds: HPLC analysis:

Phenolic reference standard for HPLC was used for analyzing phenolic compounds present in Aerva lanata extract²⁹ Waters 2487 HPLC system was used for HPLC analysis. Solution A [ 50 mM Na₂PO₃ in 10% CH₃OH of pH 3.3] and Solution B [70% CH₃OH] was used for gradient elution with decreasing concentration and elapsed time. HPLC grade methanol (10mg/mL) was used for dissolving the dried alcoholic extract and filtered through 0.22µm sterile syringe filter of which 10 µL volume was injected to the HPLC. Detection was monitored at diverse wavelength “250, 280, 320, 370 and 510nm” and Flow rate was maintained at 1 mL/min with temperature adjusted to 25 degree Celsius.

When phytochemical constituents or their metabolites reach to specific levels at their sites of action then pharmacological activity is achieved. Developing and reforming many constituents for identification of various metabolites in samples was better for safety and accuracy of herbal products. LC–MS plays significant role in detection and analysis of various active metabolites of pharmacological compounds^30.Preliminary examination of alcoholic extracts on Aerva lanata proposed the existence of compounds like “steroids, terpenoids, flavonoids, alkaloids, glycosides, sugar, carbohydrate, proteins, ash content and amino acids” with n-hexane-ethyl acetate (7.2: 2.8) as the mobile phase. Aerva lanata alcoholic extracts provided 27 types of terpenoids with 27 Rf values in the range [ 0.06 - 0.97] from the whole parts of the plant^31.

Alkaloids:

Canthin-six-one alkaloids such as“10-methoxycanthin-six-one,10-hydroxycanthin-six-one, 10-O-beta-Dglucopyranosyloxycanthin-six-one,10-hydroxycanthin-six-one[ervine], 10-beta-D-glucopyranosyloxycanthin-six-one[ervoside], 10-methoxycanthine-six one [methyl aervine], Aervine [10-hydroxycanthin-sixone], Methyl aervine [10- Methoxy canthin-six-one] and aervoside [10 beta-D- glucopyranosyl-oxyacanthine-six-one]” are the biologically active constituents present in the plant. It also contains additionally various alkaloids like “beta carboline-one-propionic acid, six methoxy beta carboline-one- propionic acid, six methoxy beta carbolinyl propionic acid [aervolanine] and aervolanine [ 3- [6 methoxy beta carbolinyl] propionic acid]”^32-34.

Flavanoids:

Various flavonoids such as “Kaempferol, quercetin, Isorhamnetin, Isorhamnetin 3-O-beta (4-P-coumaroyl-arhamnosyl(1-6) galactoside and flavanone glucoside persinol, persinosides A and B, 5, 4’-hydroxy-3,6,7-trimethoxy flavone, 5hydroxy-3,6,7,4-tetramethoxy flavone, 5-hydroxy 2’,3,5’,6,7-pentamethoxyl flavone, 3,3’,5,7-trihydroxy-4’methoxyflavone, apigenin 7-O-beta-D glucoside and 7-O-beta-D glucopyranoside.^35-37 Isorhamnetin-3-O-beta-D-glucoside, narcissin [isorhamnetin-7-O-rutinoside], feruloyl tyramine, feruloyl homo vanillyl amine, aervitrine, syringic acid, vanillic acid, ferulic acid” was been reported to be present in the exposed parts of Aerva lanata³⁷.

Others:

“Arabinose, Rhamnose, Xylose, Galactose, Glucose, Mannose and Mannitol [4.2% in A. lanata]” was isolated^38,39. Aerva lanata besides miscellaneous constituents contains “methyl grevillate, lupeol, lupeol acetate benzoic acid, ß-sitosterol acetate and tannic acid”⁴⁰.

Pharmacological studies:

Traditional Uses:

Aerva lanata shrub was utilised as demulcent and diuretic³³. Swellings can be removed by decoctions⁴¹. The Powdery part was useful in application to ulcer wounds. Aerva lanata was useful to cure urolithiasis, abdomen discomfort and internal organs inflammation⁴². The Medicinal Properties against rheumatism and kidney troubles are found to be present in flowers and root portions^43,44. Anthelminthic and analgesic property was officially recognized in the infusion extract³⁸ Antimalarial, Antivenom, Analgesic and sedative activities was present in the aerial parts of Aerva lanata ⁴⁵

Antioxidant activity:

DPPH Radicals Scavenging activity:

Aerva lanata Aqueous extracts were carried out for scavenging activity of DPPH radicals by standard protocols mentioned before^46,47. Each Methanolic extracts of “10,20,40,60,80 and 100 microgram per ml’’ dilution is mixed with 1ml of 0.2millimole per ml in methanol [ DPPH] thoroughly and was kept in incubator in dark for 40 mins at 20⁰c. UV-Vis spectrophotometer was used for measuring absorbance at 517 nm by using methanol as a blank. Scavenging activity of DPPH was measured by the ratio of difference in absorbance of control [Ac] and test [At] to the absorbance of control

% Scavenging activity = “{Ac-At} / Ac *100”

Where, Ac- control, At -test sample

Metal chelation activity:

Aerva lanata Aqueous extracts were performed for scavenging activity of DPPH radicals by standard protocols mentioned before⁴⁸.With several modifications each crude extract of “125, 250, 500 and 1 000µg/ml” dilution was mixed with 100µL of [2 mM FeCl₃]with 400µL of “5 mM ferrozoin solution” and kept to subside for several mins at room temperature (37^oC). The resultant absorbance at 562nm was measured and recorded. Metal chelating activity is measured by this formula:

percent metal chelation = [ 1- {At/Ac}] *100

Where, At is the absorbance of test, Ac is the absorbance of control.

Reduction of power activity:

Ferric Reducing antioxidant power assay was performed on aqueous extract of Aerva lanata stem for its reducing activity^49,50Various concentrations “125, 250, 500 and 1 000µg/mL of plant extract [1ml]” was mixed with 2.5 mL phosphate buffer (0.2 M at pH 6.6 of 1 % K₃Fe (CN)₆) and the whole mixture was placed in an incubator for 20 mins at 50⁰c and 10% Trichloroacetic acid (2.5mL) was placed in a mixture and placed at speed of 3000 rpm in cooling centrifuge for 10 min. The supernatant liquid portion (2.5ml) was took and allowed to be mixed with equal volume of distilled water & 0.5 mL of 0.1 % Fecl_3.UV-Visible spectrophotometer was used for measuring absorbance at 700 nm by using ascorbic acid as control. Reducing power activity was indicated by higher absorbance of the mixture.

Chronic diseases are mainly due to the reason of oxidative stress and damage. Aerva lanata leaf portion exhibits better antioxidant property. Activity was resoluted by Aqueous extract of Aerva lanata which showed “2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, metal chelating activity and reducing powder activity”^51,52.

Antimicrobial:

Gram Positive bacterial species [Bacillus, Staphylococcus] and Gram-negative bacterial species [E. coli, Shigella, Klebsiella, Candida species] are most likely to cause microbial infections and its effect is nullified by Pet ether, ethyl acetate and Carbinol extracts on the whole plant of Aerva lanata^4,53. The extract of Aerva lanata with chloroform was proved to be effective against the microbes showing significant inhibition upon both gram negative and gram-positive organisms^54-56.

Anti-urolithiasis and Diuretic:

The Ethanolic Extract of Aerva lanata “800mg/Kg acts as diuretic” when compared with control. The aqueous suspension dose [2g/kg b/w /dose/28 days] of Aerva lanatais given to CaO induced urolithiasis rats which reduced oxalate synthesizing enzymes and resulted in crystal deposition in Kidney. The observed results affirmed that Aerva lanata has a beneficial effect for kidney stones⁵⁷. Majumdar et al., stated that the ethanolic and aqueous extract on stem, root and leaf of Aerva lanata has diuretic activity significantly at 1600 mg/kg/BW dose. Flowers of Aerva lanata, 50g/L induces diuresis⁵⁸.

Immunomodulatory effect:

Aerva lanata extraction with pet ether has cytotoxic activity towards lymphoma of Daltons ascites malignant In Vitro cell lines and proliferation of lymphocyte In vitro and In vivo conditions⁵⁹. The Partially purified TLC fraction on Pet ether extract of Aerva lanata confirmed as toxic to cells of Daltons lymphoma ascites, B 16F10 cell lines, Ehrlich ascites in vitro to degrade solid tumor of cell lines in the mice model⁶⁰.

Antiparasitic:

Extracts of Aerva lanata when tested upon tapeworm and earthworm in particular with alcoholic extract of ethanol has proved efficiency against worms than Albendazole, most commonly utilized for various Parasitic infections^61,62.

Hepatoprotective activity:

Extract of Aerva lanata restored Elevated liver marker enzymes activities with beneficial antioxidant effects at 1600mg/Kg body weight⁶³. The root and leaves extract at 600mg/Kg body weight has beneficial hepatic protective activity with a dried alcoholic extract^8,60.

Acute renal failure:

Nephro protective activity was constituted to prevail in the alcoholic extract of Aerva lanata in Gentamicin besides cisplatin induced acute Kidney injury in either sex of albino rat. The elevated blood urea level and creatinine was reduced in a dose on extract [75, 150 and 300mg/kg body weight]. Minimal toxicity was found to be retained and encouraging treatment of acute injury of kidney by toxic substances such as gentamicin besides cisplatin to kidney⁶.

Antiasthmatic:

The Methanolic extract of the plant shows activity against asthma in 100mug/ml in the tracheal part of goat. The extract dose at 30mg/Kg and 60mg/Kg orally proved to be effective for asthma towards clonidine related catalepsy which inhibited the degranulation of mice in mice⁶⁴.

Antidiarrheal activity:

Hydroalcoholic extract of Aerva plant proposed to be much more effective in the treatment of diarrhea at dose 800 mg/kg in Inbred rats of albino wistar. Anti-diarrheal activity was proved to be effective in charcoal meal test. Its activity was provided by Reduction of intestinal transit which reduces the bowel movements²¹. “Charcoal meal test, castor oil induced diarrhea, enter pooling induction method by prostaglandin. The standard drug used for anti-diarrheal by castor oil method was Loperamide and Atropine was utilized for charcoal meal test⁵¹.

Antifertility activity:

Anti-fertility activity using anti-implantation, abortifacient and spermatozoa motility invitro models showed significant activity of alcoholic extract on the exposed part of Aerva lanata. The dose of Aerva lanata [200mg/Kg and 400mg/kg/BW] towards control provided pre implantation loss of 20 and 30 percent and showed zero motility of spermatozoa at 10 percent concentration in sixty seconds interval⁶⁵. The alcoholic extract of Aerva lanata proved to show the activity against fertility with anti-implantation at dose 200-400 mg/Kg⁶⁶.

Antihyperglycemic activity:

Aerva lanata leaf extract proved to produce anti hyperglycemic activity in the dose range of 100/200/400 mg proving to reduce the sugar level and improve the blood glucose level. Three doses of Oral glucose tolerance test are utilized as a confirmation for antihyperglycemic effect in alloxan induced diabetes⁶⁷. Deshmukh et all., stated that 400mg/Kg was suffice to increase glucose level at one-hour period after glucose administration. The ethanolic extract of Aerva lanata reduced sugar level in the blood of Alloxan-induced rat upto 42% in dose “375 mg/kg/BW and 48% in dose range 500 mg/kg/BW”^5,68. Treatment of diabetes with ethanolic Aerva lanata extract [375/500mg/kg] provided decline to the glucose levels in blood^68,69.

Antiulcer:

The ethanolic extract of Aerva lanata (250-500mg/kg) stem exhibited efficacious antiulcer properties when compared with standard drug⁷⁰.

Anti-inflammatory activity:

Ethanolic plant extract exhibited anti-inflammatory activity upon carrageenan induced rat^52,71. Vetrichelvan T, et all., stated that anti-inflammatory activity was observed upon carrageenan-induced paw edema in rat at dose [800 mg/kg/BW] on benzene and ethanolic extract of Aerva lanata^61,72.

Hypolipidemic activity:

The aqueous suspension of the extract was proved to show hypolipidemic activity. Ethylene glycol-induced calcium oxalate urolithiasis rats when assessed found to significantly increase the calcium oxalate and triglycerides level in serum, liver and kidney and thereby altering the levels of phospholipids “high-density lipoproteins, low-density lipoproteins and very low-density lipoproteins”. Aerva lanata aqueous suspension reverted these values to normal indicating that the suspension acted as a better hypolipidemic agent upon calcium oxalate induced urolithiasis⁷³

DNA damage inhibition efficiency:

Photolysis of H₂O₂ by UV radiation in presence of pBR322 plasmid DNA and by performing agarose gel electrophoresis with the irradiated DNA was used on aqueous extracts of Aerva lanata for measuring the DNA Damage inhibition efficiency⁷⁴.pBR322 aliquot solution [200µg/mL] was taken in three micro centrifugation tubes each containing 1µL of aliquot and 50µg of aqueous extracts is added to the tube and remaining tube is left untreated as irradiated controls. 3% H₂O₂ of 4µL was added to all the tubes and placed on UV transilluminator at 300nm for 10 min at room temperature. 4µL of tracking dye [ 0.25% bromophenol blue, 0.25% xylenol and 30% glycerol] was added after irradiation and were kept for analysis by gel electrophoresis on 1% agarose gel [ ethidium bromide] in TBE buffer of pH around 8. Untreated plasmid was run along with untreated UV irradiated plasmid DNA and aqueous treated UV- irradiated sample.

Hemolytic activity:

spectroscopic method was used for the performance of hemolytic activity on aqueous extract of Aerva lanata ⁷⁵. Erythrocyte was collected from the obtained blood of a healthy individual by repeated wash with sterilized phosphate buffer in saline solution (pH 7.2). phosphate buffer saline is used to resuspend the cells to 0.5% and 0.5ml of cell suspension is mixed with 0.5 ml of plant extracts with concentration ranging from “125, 250, 500 and 1 000µg/mL” in saline solution. The mixtures are later kept in an incubator at 37^oC for half an hour and subjected to cooling centrifugation at 1500rpm for 10 mins. UV-Visible spectrophotometer at 540nm absorbance was used to measure the hemoglobin at supernatant layer. Phosphate buffer was used as a control and percentage hemolysis was calculated using the formula

% hemolysis= At-An ×100

Ac-An

Where, At is test sample absorbance, An is minimal control absorbance, Ac is maximal control absorbance

CONCLUSION:

Aerva lanata was used as a therapeutic agent for various diseases. The main phytochemical constituents like flavonoids and alkaloids extracted from the plant might be solely responsible for its pharmacological actions. It has promising actions for urolithiasis and other such prevailing kidney related disease thereby contributing to vast research in the synthesis of new chemical entity. It was also useful in treating ulcers and other gastro related disorders thus has many beneficial effects thus contributing to better healthcare. They possess antimicrobial properties, antihyperglycemic, anti-urolithiasis, antifertility, anthelmintic activity and other pharmacological activities. Aerva lanata was used to synthesize various drugs and thus acts as an important source for new therapeutic agents.

ACKNOWLEDGEMENT:

This work was supported by the respected Dean of S.R.M. College of Pharmacy, SRM Institute of

Science and Technology, Kattankulathur.

CONFLICT OF INTEREST:

This article does not contain any conflict of interest.

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Received on 21.08.2019 Modified on 21.10.2019

Research J. Pharm. and Tech 2020; 13(3):1580-1586.

DOI: 10.5958/0974-360X.2020.00286.3