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B.J.M. Reddy et al. J Syn Natu Chem, 2017;2(4):21-30

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World Inventia Publishers

Journal of Synthetic and Natural Chemistry http://www.worldinventiapublishers.com/JSNC/

Vol. 2, Issue 4, 2017

Research Article

ISOLATION, SPECTRAL CHARACTERIZATION AND BIOLOGICAL EVOLUTION OF PERIPHERAL ANALGESIC ACTIVITY ON AERIAL PARTS OF AERVA LANATA LINN

Dr. B. Jagan Mohan Reddy 1*, B. Madhavi Latha 2, G. Geetha Rani 2, Dr. A. Madhukar 3

* 1 Adikavi Nannaya University, Rajah Rajah Narendhra Nagar, Rajahmundry, A.P - 533296, INDIA. 2 Department of Pharmacy, Brilliant Group of Technical Institutions, Abdullapurmet, Hyderabad, Telangana, INDIA.

3 Associate Professor, Avanthi Institute of Pharmaceutical Sciences, Gunthapally, R.R. Dist., Hyderabad, Telangana, INDIA.

Received on: 05-11-2017; Revised and Accepted on: 12-12-2017

ABSTRACT

The aim of the present study was to evaluate the Analgesic Activity on Aerial Parts of Aerva Lanata Linn in animal models. The analgesic

activity of the extract was evaluated for its central and peripheral pharmacological actions by using Acetic acid-induced writhing method and Tail

immersion method respectively. Chemical investigation of the aerial parts of Aerva lanata Linn lead to the isolation of compounds namely Stigmasterol,

campesterol, aervine, Betulin. The constituents isolated and characterized from the aerial parts of the plant Aerva lanata Linn can be categorized under

the sterols, alkaloids and triterpenoids. From the petroleum ether (60 - 80˚) extract sterols like Stigmasterol, campesterol, From chloroform extract

alkaloids like aervine were isolated and characterized by spectral data (1HNMR, 13CNMR & MASS). These compounds were isolated for the first time

from the aerial parts of Aerva lanata Linn. The study was carried out in two different dose levels of 200 and 400 mg kg-1 orally. The Aerva lanata

produce Minimum inhibition dose of 200 mg kg-1. Aerva lanata at the dose of 400 mg kg-1 showed maximum inhibition. In Tail immersion method all

these extracts and isolated compounds shows significant (P < 0.01) analgesic activity at 3 hour and marked analgesic activity at 2 hour as compared to

control. The pharmacological screening of the extract showed significant (p < 0.01-0.05) dose-dependent Analgesic activity when compared with

reference standard. The presence of flavonoids might be responsible for these activities and which are probably mediated via inhibition of various

autocoids formation and release.

KEYWORDS: Stigmasterol, Campesterol, Aervine, Analgesic Activity, Aerva lanata Linn.

INTRODUCTION

Aerva lanata (Linn) Juss. ex Schult of Amaranthaceae family

is commonly identified and known as Gorakshaganja in Ayurveda system of medicine. It is considered as one among the few botanical sources of Pashanabheda. The plant is extensively used in urinary disorders like Ashmari (Urinary calculi), Mootrakrichra (Dysuria), Mootravikara etc by most of the Ayurveda and Siddha practitioners in southern India, in the name of Pashanabheda. As the plant bears almost all the properties similar to that of the original source of Pashanabheda [1]. Few synonyms mentioned for this plant are: Aadanapaki, Shatakabheda [2], Valliyaka, Tripatra, Krsnavalli, Prayanika [3]. It is identified as Shwethashelaa, Astmabayda, Bhadra in Sanskrit [4], Gorakhaganja, Gorakhabooti, Kapurijadi in Hindi [2], Mountain knot grass in English [5], Bilihindee soppu, Vibhoothikasa, Pashanabhedi in Kannada, Chirupoolaiin Tamil, Pindikoora/ Kondapindi in Telgu, Cherula in Malayalam [4], Karur-madhurain Marathi, Buikallan in Punjabi, Paunsia in Odiya. And it is one of the plants included in Dasapushpam, the ten sacred flowers of Kerala.

Plants have been used in traditional medicines for thousands of years. Medicinal plants as a group comprise approximately 8000 species and account for about 50% of all the higher flowering plant

*Corresponding author: Dr. B. Jagan Mohan Reddy Adikavi Nannaya University, Rajah Rajah Narendhra Nagar, Rajahmundry, A.P - 533296, INDIA. * E-Mail: [email protected]

species in India. The knowledge of medicinal plants has been accumulated in the course of many centuries based on different medicinal systems such as Ayurveda, Unani and Siddha. In large number of countries human population depends on medicinal plants for treating various illnesses as well as source of livelihood. One of the main advantages of using medicinal plants is that, these do not produce or cause any side effects when compared with synthetic drugs, because medicinal plants have high content of antioxidant compounds. This gives protective effects against diseases without reducing their therapeutic efficacy [1]. The Indian traditional system of medicine, especially Ayurveda has put forward a number of therapeutic claims on plant drugs. However it is important to conduct thorough investigation of many traditionally used medicinal plants with reference to modern system of medicine [2]. The World Health Organization estimated that 80% of the population of developing countries relies on traditional medicines, mostly plant drugs for their primary health care needs [2-5]. Nowadays herbal drugs have become world important objects, with both medicinal and economic implications.

A regular and wide spread use of herbs throughout the world has increased serious concerns over their quality, safety and efficacy [6]. These are because of the nature of herbal ingredients present there, which are complex mixtures of different secondary metabolites that can vary considerably, depending on environmental and genetic factors. These complex positions of quality aspects of herbal drugs are complicated by the use of combination of herbal ingredients. The standardization methods of medicinal plants and their extracts have great important [7]. Like this the reliable scientific efficacy and quality aspects of Aerva lanata has to be maintained.

Plant description: Aerva lanata belongs to the family of Amaranthaceae. It is one of the important medicinal plants have ever grown throughout the plains of India. Aerva lanata is found to be an

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erect or prostrate herbaceous weed that is common throughout the hotter parts of India especially all over the plains, this extends up to an altitude of 3000m. It is also be present in Sri Lanka, Arabia, Egypt, tropical Africa, Java and Philippines. In India, it spreads in the states of Tamil Nadu, Andra Pradesh and Karnataka. Aerva lanata had been used in the Indian folk medicine for the treatment of diabetes mellitus, urinary calculi, hematesis, bronchitis, nasal bleeding, cough, scorpion stings, fractures, spermatorrhea, to clear uterus after delivery and also to prevent lactation [8].

Medicinal properties: The plant Aerva lanata is diuretic and used in lithiasis. The

roots are demulcent, diuretic, and useful in strangury, cure kidney stones [2]. The root has a camphor like aroma and medicinally important. Decoctions of the flowers are used to cure stones in any parts of the stomach [10]. The roots are also used in the treatment of headache. The plant is regarded as a demulcent on the Malabar Coast. It has valued for cough in Ceylon and also as a vermifuge for children. The Meena tribals of the Sawaimadhopur district of Rajasthan orally used to give the juice of the roots of Aerva lanata to patients who are suffering from liver congestion, jaundice, biliousness and dyspepsia. They also give decoction of the whole plant to cure pneumonia, typhoid and other prolonged fevers [12]. Traditionally, leaves of Aerva lanata are used as sap for eye-complaints; an infusion is given to cure diarrhoea and kidney stone; and the root is used in snake bite treatment. The decoction of leaf is used as gargle for treating sore throat and it is also used in various complex treatments against guineaworm [13]. In addition to the traditional uses, the plant is reported for a number of pharmacological activities viz., anthelmintic, demulcent, anti-inflammatory, diuretic, expectorant, hepatoprotective and nephroprotective , anti-diabetic, anti-hyperglycemic, anti-microbial, cytotoxic , urolithiatic , hypoglycemic, anti-hyperlipidemic , anti-parasitic and anti -helmintic activities [11, 14], Antimicrobial and Antioxidant activity of Aerva lanata, Diuretic activity of Aerva lanata: [19], The anti-inflammatory activity [28], Antidiarrhoeal activity [15], Antidiabetic effect of Aerva lanata [16], Cardio protective effect of Aerva lanata: Cardioprotective effect [17], Antihepatatic activity [18].

MATERIALS AND METHODS

Biological History: Botanical name - Aerva Lanata Family - Amaranthaceae

Habitat - Herb Ayurvedic name - paashaanabheda, Gorakshayanjaa, Aadaanpaahi, Shatkabhedi Bengali name - Chaya Hindi name - kapurijadi, Gorakhabumdi [9].

Fig. 1: Geographical appearance of Aerva Lanata

Experimental Procedure: 1. Collection and Authentification of Aerva lanata Linn:

The whole plant, Aerva lanata Linn was collected from land scape of local areas of Hyderabad. It was authenticated by Dr. B. Bhadraiah, HOD, Dept of Botany, Osmania University, Hyderabad. Voucher specimens are kept at the Mall Reddy College of Pharmacy, Dhullapally, Hyderabad, Telangana, India.

2. Preparation of different extracts from aerial parts of Aerva lanata Linn:

The shade dried aerial parts of Aerva lanata were reduced to fine powder and around 5.0 kg were subjected to continuous successive extractions with different solvents of increasing polarity like Petroleum ether (60-80o), chloroform, ethylacetate and ethanol (70%) into 15 batches of each 250-300 gm each in a soxhlet extractor. These extracts were filtered and concentrated to dryness in rotavapour (Buchi labortechnic,Flawal ,Switzerland) These dried extracts were stored in desicator.

After drying the respective extracts were weighed and percentage yield were recorded in

Table No. 1: Preparation of different extracts from aerial parts of Aerva lanata Linn

S. No. Extract Nature of the extract Color of the extract weight(gm) Percentage yield (%)

1 Petroleum ether (80o) extract

Sticky mass Light green colour 44 0.88%

2 Chloroform extract Sticky mass Dark green colour 40 0.8%

3 Ethyl acetate extract Sticky mass Light Yellow colour 39 0.78%

4 Ethanol (70%) extract Sticky mass Dark green colour 50 1%

Preliminary Phytochemical analysis: 1. Preliminary Qualitative Chemical Investigation:

All the above different extracts were subjected to preliminary chemical investigation. The following procedures were adopted to test for the presence of various chemical constituents in the extracts.

2. Test for Sterols: a. Salkowaski test:

2mg of dry extract of flower was shaken with chloroform. To the chloroform layer sulphuric acid was added slowly by the sides of the test tube. Formation of red color indicates the presence of sterols.

b. Liberman-Burchard’s test: 2 mg of dry extract of flower was dissolved in acetic anhydride,

heated to boiling, cooled and then 1 ml of concentrated sulphuric acid was added along the sides of the test tube. Formation of green color indicates the presence of steroids.

3. Test for Flavonoids: a. Shinodas’s test:

In a test tube containing 0.5 ml of the extract of flower, 10 drops of dilute hydrochloride acid was added, followed by a piece of magnesium. Formation of pink or reddish or brown color indicates the presence of flavonoids.

4. Test for Triterpenoids: a. Liberman-Burchard’s test:

2 mg of dry extract of flower was dissolved in acetic anhydride, heated to boiling, cooled and then 1 ml of concentrated sulphuric acid was added along the sides of the test tube. Formation of violet colored ring indicates the presence of triterpenoids.

5. Test for Saponins: a. Foam test:

In a test tube containing about 5 ml of extract of flower a drop of sodium bicarbonate solution was added. The test tube was shaken vigorously and left for 3 minutes. Formation of honeycomb like foam/froth indicates the presence the presence of saponins.




6. Test for Carbohydrates: a. Molish’s test:

In a test tube containing 2 ml of the extract of flower, 2 drops of freshly prepared 20 % alcoholic solution of α–naphthol was added. 2 ml of concentrated sulphuric acid was added, so as to form a layer below the mixture. Red-violet ring appeared indicating the presence of carbohydrates, which disappeared on the addition of excess of alkali.

b. Benedict’s test: To 2 ml of the extract of flower, 5 ml of Benedict’s solution was

added and boiled for 5 minutes. Formation of brick red colored precipitate indicates the presence of carbohydrates.

c. Fehling’s test: To 2 ml of extract of flower, 1 ml of mixture of equal parts of

Fehling’s solution A and B were added and boiled for few minutes. Formation of red or brick red colored precipitate indicates the presence of reducing sugar.

7. Tests for Alkaloids: a. Mayer’s test:

To a few drops of the Mayer’s reagent, 2 mg of extract of flower was added. Formation of white or pale yellow or buff colored precipitate indicates the presence of alkaloids.

b. Wagner’s test: 2 mg of extract of flower was acidified with 1.5 % v/v of

hydrochloric acid and a few drops of Wagner’s reagent were added. A yellow or brown colored precipitate indicates the presence of the alkaloids.

c. Hager’s test: To 2 mg of the extract of flower taken in a test tube, a few drops of Hager’s reagent were added. Formation of yellow colored precipitate confirms the presence of alkaloids.

d. Dragendroff’s test: To 2 mg of extract of flower, 5 ml of distilled water was

added; 2 M Hydrochloric acid was added until an acid reaction occurs. To this 1 ml of Dragendroff’s reagent was added. Formation of orange or orange red colored precipitate indicates the presence of alkaloids.

8. Test for Tannins: To 1-2 ml of the extract of flower, few drops of 5 % w/v FeCl3

solution was added. A green colored indicates the presence of gallotannins, while brown color indicates the presence of pseudo tannins.

9. Test for Proteins:

a. Millon’s test: 1 ml of ethanolic extract of flower was dissolved in 1 ml of

distilled water and 5-6 drops of Millon’s reagent were added. Formation of white colored precipitate which turns red on heating indicates the presence of proteins.

b. Biuret test: To 1 ml of hot extract of flower, 5-8 drops of 10 % w/v

sodium hydroxide solution was added, followed by 1 or 2 drops 3 % w/v copper sulphate solution were added. Formation of a violet red color indicates the presence of proteins.

10. Test for Resins: 1 ml of extract of flower was dissolved in acetone and

solution was poured in distilled water. Turbidity indicates the presence of resins.

11. Test for Starch: 0.01 g of iodine and 0.075 g of potassium iodide were

dissolved in 5 ml of distilled water and 2-3 ml of extract of flower was added. Formation of blue color indicates the presence of starch.

12. Test for Glycosides: a. Legal test.

Flower extract is dissolved in pyridine, sodium nitroprusside solution is added to it and made alkaline. Pink red color is produced.

b. Baljet test: To the flower extract, sodium picrate solution is added;

yellow to orange color is produced.

c. Borntrager test: Few ml of dil. sulphuric acid was added to the test solution. Boil,

filter and extracted the filtrate with ether or chloroform. The organic layer is separated to which ammonia is added; pink, red or violet color is produced in organic layer.

d. Keller Killani test: Sample was dissolved in acetic acid containing trace of ferric

chloride and transferred to the surface of concentrated sulphuric acid. At the junction of liquid reddish brown color was produced which gradually becomes blue.

The preliminary qualitative chemical investigation of Chlorofom extracts, ethyl acetate and ethanolic extracts of flowers of Aerva lanata was showed in Table-1.

The results of qualitative chemical investigation of Chlorofom, ethyl acetate and ethanolic extracts of flowers of Aerva lanata indicated the presence of compounds listed in Table-2

Table No. 2: Preliminary Qualitative Chemical Investigation tests

Chemical Test Petroleum ether Extract Chloroform Extract Ethyl Acetate Extract Ethanol Extract (70%)

Sterols + - - +

Triterpenoids + - - +

Flavonoids - - + +

Saponins - - - +

Carbohydrates - - - +

Alkaloids - + - +

Tannins - - + +

Proteins - - - -

Glycosides - - - +

RESULTS AND DISCUSSION

Isolation from Petroleum ether extract: The concentrated petroleum ether extract (30 g) was

dissolved in n-Hexane (20 ml) and chromatographed through a column of Silica gel 60-120 mesh LR (diam. 4 cm X length 45 cm). The column being successively eluted with n-Hexane: ethyl acetate in graded mixture i.e. 98:2, 96:4, 94:6, 92:8,………up to 100 %. From above

elution’s, four different fractions were collected (i.e. fraction A, B, C and D).

Characterization of Compound1 (AN-1): Fraction A was eluted from n-hexane: ethyl acetate (98:2), resulted gives a single compound which was confirmed by TLC (n-hexane: ethyl acetate, 8.5:1.5). It was recrystallized with acetone. The product was designated as compound AN-1




Spectral Analysis: IR (KBr): 3430.47 cm-1

(O-H, stretching); 2921.03 & 28151.74 cm-1

(CH2,

asymmetric & Symmetric stretching); 1736.32 cm-1 (non-conjugated C=C

stretching); 1460.85 cm-1 (CH2,

bending); 1377.23 cm-1 (alkene, CH3

bending); 1216.59 cm-1 (C-O, stretching). 1HNMR (δ Values ppm): 5.4 (m, 1H, olefinic protons, H-6); 4.0 (s, 1H, CH-OH, vinylic protons); 2.82 (m, 1H, vinylic protons, H-3); 2.38 to 1.2 (m, 18H, 19 X CH2 & 1H of CH3

group); 0.9 to 0.7 (m, 18H, 6 X CH3 group). 13C NMR (δ Values ppm): 37.8 (C-1 & 10), 31.9 (C-2), 70.1 (C-3), 41.3 (C-4), 141.9 (C-5), 124.3 (C-6), 32.1 (C-7), 30.1 (C-8), 37.9 (C-9), 21.1 (C-11), 42.3 (C-12), 46.1 (C-13), 58.6 (C-14), 24.1 (C-15), 27.2 (C-16), 62.0 (C-17), 11.3 (C-18), 22.9 (C-19), 40.9 (C-20), 20.5 (C-21), 135.3 (C-22), 130.1 (C-23), 59.0 (C-24), 30.9 (C-25).

Mass spectra: IMS (m/z)(%) Rel. Intensity: 4 10 [M +2], [C29 H46O], (15 %), 396 (23 %), 329 (8 %), 303 (12 %), 271 (13 %), 255 [M+, side chain,H2O] ( 24 %), 159 (4 %), 83 ( 32 %).

Mass Spectrum (MS) showed [M+] at m/z 410, corresponding to the molecular formula (C29H46O). It was found to be identical with “Stigmasterol” on comparison with authentical sample (mixed M.P. CO-TLC and super impossible IR).

Fig. 2: Structure of Stigmasterol

From the M. P, I. R, 1HNMR and Mass spectra compound AN-1 was ascertained as “Stigmasterol”

Characterization of Compound 2 (AN-2): Fraction B was eluted from n-hexane: ethyl acetate (70:30), resulted gives a single compound which was confirmed by TLC (n-hexane: ethyl acetate, 7:3). This elute was collected and concentrated to about 10 ml crude residue substance obtained. The product was designated as compound AN-2.

1HNMR (δ Values ppm): 5.4 (s, 1H, olefinic proton); 3.4 (s, 1H, OH group); 3.28 (m, 1H, vinylic proton H-3); 2.4 to 1.0 (m, 20H 10 X CH2 protons); 1.6 (s, CH2 protons); 0.9 to 0.7 (m, 6 X CH3 group H-18, 19, 21, 26, 27 & 28); 0.8 (m, 9H, 3 X CH3 group H-30, 32 & 33). 13C NMR (δ Values ppm): 33.6 (C-1 & C-12), 34.1 (C-2), 77.0 (C-3), 62.1 (C-4), 130.1 (C-5), 117.8 (C-6), 32.0 (C-7), 31.6 (C-8), 68.4 (C-9, C-14 & C-17), 37.2 (C-10 & C-20), 24.3 (C-11), 32.4 (C-13 & C-23), 25.1 (C-15), 25.3 (C-16), 13.9 (C-18), 14.5 (C-19), 29.5 (C-21), 30.1 (C-22), 39.1 (C-24), 32.6 (C-25), 28.1 (C-26 & 27), 15.3 (C-28).

Mass spectra: EIMS (m/z) (%) Rel. Intensity: 395 (M+) (33 %), 382 (11 %), 367 (5 %), 358 (14 %), 315 (25 %), 289 (11 %), 230 (16 %), 213 (4 %)

Fig. 3: Structure of Campesterol

Mass Spectrum (MS) showed [M+] at m/z 395, corresponding to the molecular formula (C29H35O). It was found to be identical with “Campesterol” on comparison with authentical sample (mixed M.P. CO-TLC and super impossible IR.

Characterization of Compound 3 (AN-3): Fraction C was eluted from n-hexane: ethyl acetate (80:20), resulted gives a single compound which was confirmed by TLC (n-hexane: ethyl acetate, 7:3). On evaporation gave a crystalline powder. This elute was collected and concentrated to about 10 ml crude residue substance obtained. It was recrystalised with acetone. The product was designated as compound AN-3.

Spectral Analysis: 1HNMR (δ Values ppm): 7.1 to 5.98 (m, 3H, Aromatic proton); 6.4 to 6.2 (m, 2H, CH group C- 4, 5); 5.9 (d, 1H, C-2); 5.35 (d, 1H, C-1); 4.35 (m, 1H, adjacent to N-H group); 2.19 (s, 1H, NH group). 13C NMR (δ Values ppm): 110.1 (C-1), 135.1 (C-2 & C-4), 62.1 (C-3), 130.9 (C-5), 46.1 (C-6), C-N (C-7), 128.2 (C-7a & C-12), 117.1 (C-8), 113.5 (C-9), 152.5 (C-10), 99.8 (C-11), 104.1 (C-13), 132.1(C-14).

Mass spectra: EIMS (m/z) (%) Rel. Intensity: 232 [M+2, C14H6N2O2], (36 %), 222 (21 %), 184 (3 %), 133 (17 %), 117 (19 %), 94 (46 %), 83 (54 %).

Mass Spectrum (MS) showed [M+] at m/z 232, corresponding to the molecular formula (C14H6N2O2). It was found to be identical with “Aervine” on comparison with authentical sample (mixed M.P. CO-TLC and super impossible IR).

Fig. 4: Structure of Aervine

From the M. P, I. R, 1HNMR and Mass spectra compound AN-3 was ascertained as “Aervine”.

Characterization of Compound 4 (AL-4): Fraction C was eluted from chloroform: methanol (59:41), resulted gives a single compound which was confirmed by TLC chloroform: methanol, (7:3). This elute was collected and concentrated to about 10 ml crude residue substance obtained. It was recrystalised with acetone. The product was designated as compound AN-4.

1HNMR (δ Values ppm): 4.9 to 4.63 (s, 2H, C=CH2 group C-29); 3.70 to 3.58 (s, 2H, OH group); 3.25 (d, 2H,CH2 group H-28); 2.84 (m, 1H, H-3); 2.30 (m, 3H, CH3-C=CH2, H-30); 2.0 to 1.05 (m, 25H for CH2 & CH protons); 0.90 (m, 15H, 5 X CH3 group).

13C NMR (δ Values ppm): 39.2

(C-1 ), 27.1(C-2), 78.0 (C-3), 37.3 (C-4 & 8), 55.4( C-5), 19.5 (C-6), 32.9 (C-7), 50.5 (C-9 & 14), 38.5 (C-10), 18.5 (C-11), 36.2 (C-12), 34.1 (C-13), 31.7 (C-15), 32.3 (C-16), 35.6 (C-17), 40.4 (C-18 & 19), 150.1 (C-20), 29.2 (C-21,23 &24), 2.6 (C-22), 19.5 (C-25 & 27), 24.5 (C-26), 76.5 (C-28), 100.1 (C-29), 27.1 (C-30).

Mass spectra: EIMS (m/z) (%) Rel. Intensity: 440 [M+1, C30H50O2], (5 %), 426 (9 %), 412 (13 %), 386 (8 %), 318 (18 %), 304 (9 %), 274 (15%), 220 (17 %), 152 (35 %), 138 (22 %), 83(30 %).

Mass Spectrum (MS) showed [M +1] at m/z 440, corresponding to the molecular formula (C30H50O2) It was found to be identical with “ Betulin: on comparison with authentical sample (mixed M.P. CO-TLC and super impossible IR).




Fig. 5: Structure of Betulin

Table No. 3: Spectral analysis of compound – AN-1

Compound 1H NMR MASS 13C NMR

Reddish brown colour sticky compound Rf: 0.85 (Solvent system: CHCl3: MeOH, 9:1) M. P: 162- 165 oC.

5.4 (m, 1H, olefinic protons, H-6), 4.0 (s,1H, CH-OH, vinylic protons), 2.82 (m, 1H, vinylic protons, H-3), 2.38 to 1.2 (m,18H, 19 X CH2 & 1H of CH3 group), 0.9 to 0.7 (m, 18H, 6 X CH3 group)

412 [M+1] (100%), 397 (12%), 256 (50%), 213 (48%), 83 (37%), 43 (35%)

37.8 (C-1 & 10), 31.9 (C-2), 70.1 (C-3), 41.3 (C-4), 141.9 (C-5), 124.3 (C-6), 32.1 (C-7), 30.1 (C-8), 37.9 (C-9), 21.1 (C-11), 42.3 (C-12), 46.1 (C-13), 58.6 (C-14), 24.1 (C-15), 27.2 (C-16), 62.0 (C-17), 11.3 (C-18), 22.9 (C-19), 40.9 (C-20), 20.5 (C-21), 135.3 (C-22), 130.1 (C-23), 59.0 (C-24), 30.9 (C-25).

Fig. 6: 1H spectra of the Stigmasterol

Fig. 7: 13C spectra of the Stigmasterol




Fig. 8: MASS Spectra of the Stigmasterol

Table No. 4: Spectral analysis of compound – PEE2

Compound H1 NMR 13C NMR MASS

Reddish yellow colour sticky compound Rf: 0.81 (Solvent system: chloroform: MeOH, 9:1) M. P: 156 - 158 oC.

5.4 (s, 1H, olefinic proton), 3.4 (s, 1H, OH group), 3.28 (m, 1H, vinylic proton H-3), 2.4 to 1.0 (m, 20H 10 X CH2 protons), 1.6 (s, CH2 protons), 0.9 to 0.7 (m, 6 X CH3 group H-18, 19, 21, 26, 27 & 28), 0.8 (m, 9H, 3 X CH3 group H-30, 32 & 33)

33.6 (C-1 & C-12), 34.1 (C-2), 77.0 (C-3), 62.1 (C-4), 130.1 (C-5), 117.8 (C-6), 32.0 (C-7), 31.6 (C-8), 68.4 (C-9, C-14 & C-17), 37.2 (C-10 & C-20), 24.3 (C-11), 32.4 (C-13 & C-23), 25.1 (C-15), 25.3 (C-16), 13.9 (C-18), 14.5 (C-19), 29.5 (C-21), 30.1 (C-22), 39.1 (C-24), 32.6 (C-25), 28.1 (C-26 & 27), 15.3 (C-28).

395 (M+) (33 %), 382 (11 %), 367 (5 %), 358 (14 %), 315 (25 %), 289 (11 %), 230 (16 %), 213 (4 %)

Fig. 9: 1H spectra of the Compeasterol

Fig. 10: 13C spectra of the Compeasterol




Table No. 5: Spectral analysis of compound – EAE1

Compound IR spectra H1 NMR 13C NMR MASS

Rf: 0.45 (chloroform: EtOAc; 8:2) 4. Coloured spot: violet colour spot. 5. M. P: 263- 267 °C.

3420.54 cm-1 (O-H stretching in CH2 & CH3 group), 3386.08 cm-1 (N-H stretching), 2920.54 & 2851.08 cm-1 (CH2 stretching) 1741.59 cm-1 (C=C stretching), 1461.86 cm-1 (C-H deformation in CH3).

7.1 to 5.98 (m, 3H, Aromatic proton), 6.4 to 6.2 (m, 2H, CH group C- 4,5), 5.9 (d, 1H, C-2), 5.35 (d, 1H, C-1), 4.35 (m, 1H, adjacent to N-H group), 2.19 (s, 1H, NH group)

110.1 (C-1), 135.1 (C-2 & C-4), 62.1 (C-3), 130.9 (C-5), 46.1 (C-6), C-N (C-7), 128.2 (C-7a & C-12), 117.1 (C-8), 113.5 (C-9), 152.5 (C-10), 99.8 (C-11), 104.1 (C-13), 132.1(C-14).

232 [M+2, C14H6N2O2], (36 %), 222 (21 %), 184 (3 %), 133 (17 %), 117 (19 %), 94 (46 %), 83 (54 %).

Fig. 11: 1H spectra of the Aervine

Fig. 12: 13C spectra of the of Aervine

Fig. 13: MASS spectra of the Aervine




Table No. 6: Spectral analysis of compound – AL-4

Fig. 14: 1H spectra of the Betulin

Fig. 15: 13C spectra of the Betulin

Fig. 16: MASS spectra of the Betulin

Compound IR spectra H1 NMR 13C NMR MASS

Betulin Pale green colour sticky compound Rf: 0.76 (Solvent system: chloroform:MeOH, 9:1) M. P: 256-257 oC.

3523.64 cm-1 (-OH, str) 2984.46 cm-1 (C-H, Str) 1233.31 cm-1 (C=O, str) 1737.09 cm-1 (C=C, str) 1462.61 cm-1 (CH2, str) 1463.43 cm-1 (C-H, def) 722.09 cm-1 (C=C, def)

4.9 to 4.63 (s, 2H, C=CH2 group C-29), 3.70 to 3.58 (s, 2H, OH group), 3.25 (d, 2H,CH2 group H-28), 2.84 (m, 1H, H-3), 2.30 (m, 3H, CH3-C=CH2, H-30), 2.0 to 1.05 (m, 25H for CH2 & CH protons), 0.90 (m, 15H, 5 X CH3 group)

39.2 (C-1 ), 27.1(C-2), 78.0 (C-3), 37.3 (C-4 & 8), 55.4( C-5), 19.5 (C-6), 32.9 (C-7), 50.5 (C-9 & 14), 38.5 (C-10), 18.5 (C-11), 36.2 (C-12), 34.1 (C-13), 31.7 (C-15), 32.3 (C-16), 35.6 (C-17), 40.4 (C-18 & 19), 150.1 (C-20), 29.2 (C-21,23 &24), 2.6 (C-22), 19.5 (C-25 & 27), 24.5 (C-26), 76.5 (C-28), 100.1 (C-29), 27.1 (C-30).

440 [M+1, C30H50 O2], (5 %), 426 (9 %), 412 (13 %), 386 (8 %), 318 (18 %), 304 (9 %), 274 (15%), 220 (17 %), 152 (35 %), 138 (22 %), 83(30 %).




Biological evaluation:

Table No. 7: Treatment of peripheral Analgesic activity to different groups

Treatment Dose(mg/kg) Number of Writhing’s Percentage Inhibition (%)

Control ---- 82.20 ± 2.01 ----

Aspirin 100 25.50± 1.74** 68.97%

Petroleum ether Extract 200 42.40 ± 1.03** 48.41%

400 39.20 ± 0.66** 52.31%

Chloroform Extract 200 61.40 ± 1.03 25.30%

400 59.20 ± 1.05 27.90%

Ethyl acetate Extract 200 45.80 ± 1.08* 44.28%

400 38.40 ± 1.08** 53.28%

Ethanol (70%) Extract 200 33.20 ± 1.08** 59.61%

400 29.60 ± 1.56** 63.99%

Isolated compound II ( Aervine)

200 53.80. ± 0.80 34.54%

400 41.40± 1.80* 49.63%

Isolated compound III (Betulin)

200 30.38 ± 0.76** 62.08%

400 27.45 ± 0.81** 66.14% Statistical analysis was done by ANOVA followed by Dunnet’s test; All the values are expressed as mean ± SEM; *P < 0.05, **P < 0.01. When compared to control.

The results of peripheral analgesic activity was assessed by acetic acid induced-writhing test, which showed significant (P < 0.01) (Table No. 7) suppression of writhing by petroleum ether, ethyl acetate, extracts and isolated compounds (Daucosterol and Lupeol) (at a doses of 200 & 400 mg/kg b.w) exhibited significant (p <0.01) analgesic activity as compared to aspirin induced control. The remaining

chloroform extract and isolated compound (Aervine) (at a doses of 200 and 400 mg/kg body weight) exhibited marked (P<0.05) analgesic activity as compared to aspirin induced control It was also observed that onset of writhing was delayed and duration of writhing was shortened in a dose dependent manner, suggesting a peripheral analgesic activity.

Table No. 8: Treatment of central Analgesic activity to different groups

GROUPS TREATMENT

Group 1st Served as control receiving 0.6 % w/v of sodium CMC suspension orally

Group 2nd Served as a positive control and received acetyl salicylic acid (aspirin) standard drug (150 mg/kg) orally, as suspension in sodium CMC (0.5 ml of 0.6 % w/v Solution

Group 3rd & 4th Animals were treated with (200 & 400 mg/kg b.w) of petroleum extract of A. lanata (0.5 ml of 0.6 % w/v solution, orally) respectively.

Group 5th & 6th Animals were treated with (200 & 400 mg/kg b.w) of chloroform extract of A.lanata (0.5 ml of 0.6 %w/v solution, orally) respectively

Group 7rd & 8th Animals were treated with (200 & 400 mg/kg b.w) of ethyl acetate extraction of A.lanata (0.5 ml of 0.6 %w/v solution, orally) respectively

Group 9th & 10th Animals were treated with (200 & 400 mg/kg b.w) of ethanolic extraction (70 %) of A.lanata (0.5 ml of 0.6 %w/v solution, orally) respectively

Group 10th & 11th Animals were treated with (200 & 400 mg/kg b.w) of AL-9 (daucosterol) (0.5 ml of 0.6 %w/v solution, orally) respectively

Group 11th & 12th Animals were treated with (200 & 400 mg/kg b.w) of AL-1 (aervine) (0.5 ml of 0.6 %w/v solution, orally) respectively

Group 13th & 14th Animals were treated with (200 & 400 mg/kg b.w) of AL-7 ( lupeol) (0.5 ml of 0.6 %w/v solution, orally) respectively.

Table No. 9: Treatment of central Analgesic activity by different dose of extraction

Treatment Dose (mg/kg) 0 sec 30 sec 60 sec (1hr) 2 hr 3 hr

Control ---- 4.3 ± 0.6 5.5 ± 0.14 6.0 ± 0.21 7.0 ± 0.3 6.9 ± 0.23

Aspirin 100 6.1± 0.22 8.2 ± 0.11 9.0 ± 0.8* 23.2 ± 2.0** 34.3 ± 7.0**

Petroleum ether Extract

200 4.6 ± 0.22 4.7 ± 0.12 5.3 ± 0.66 16.2 ± 0.44* 25.8 ± 0.23**

400 5.0 ± 0.13 5.3 ± 0.33 6.0 ± 0.42 16.9 ± 0.90* 30.4 ± 0.24**

Chloroform Extract

200 4.0 ± 0.23 4.6 ± 0.43 5.3 ± 0.22 16.2 ± 0.23* 25.6 ± 0.31**

400 4.8 ± 0.33 5.3 ± 0.23 7.0 ± 0.56 16.8 ± 0.34* 29.5 ± 0.28**

Ethyl acetate Extract

200 4.8 ± 0.88 5.0 ± 0.23 5.8 ± 0.28 16.5 ± 0.77* 27.1± 0.32**

400 5.2 ± 0.23 6.1 ± 0.99 6.9 ± 0.12 18.8 ± 0.43* 31.9 ± 0.32**

Ethanol (70%) Extract

200 4.9 ± 0.58 5.0 ± 0.11 5.8 ± 0.18 16.8 ± 0.43* 27.9 ± 0.65**

400 5.4 ± 0.13 6.2 ± 0.54 7.2 ± 0.38 19.0 ± 0.31* 32.0 ± 0.44**

Isolated compound II ( Aervine)

200 4.6 ± 0.48 4.9 ± 0.75 5.9 ± 0.32 16.4 ± 0.73* 26.5 ± 0.58**

400 5.1 ± 0.31 5.8 ± 0.85 6.2 ± 0.53 17.0 ± 0.42* 30.3 ± 0.08**

Isolated compound III (Betulin)

200 5.2 ± 0.72 5.9 ± 0.38 6.4 ± 0.43 17.7 ± 0.28* 28.9 ± 0.75**

400 5.8 ± 0.66 6.7 ± 0.39 7.8 ± 0.61 19.5 ± 0.34 32.5 ± 0.80** Values are represents mean ± SEM; **P < 0.01 as compared to control indicates more significant analgesic activity; *P < 0.05 as compared to control indicates moderate

significant activity.




The results of central analgesic activity was assessed by tail immersion test, which showed that all these extracts and isolated compounds shows significant (P < 0.01) analgesic activity at 3 hour and marked analgesic activity at 2 hour as compared to control (Table No. 9).

Analgesic activity: All these extracts and isolated compounds were subjected to

analgesic activity by using two methods. In acetic method petroleum ether, ethyl acetate, extracts and isolated compounds (Daucosterol and Lupeol) (at a doses of 200 & 400 mg/kg b.w) exhibited significant (p <0.01) analgesic activity whereas chloroform extract and isolated compound (Aervine) (at a doses of 200 and 400 mg/kg body weight) exhibited marked (P<0.05) analgesic activity as compared to aspirin induced control.

In Tail immersion method all these extracts and isolated compounds shows significant (P < 0.01)analgesic activity at 3 hour and marked analgesic activity at 2 hour as compared to control.

CONCLUSION

Chemical investigation of the aerial parts of Aerva lanata

Linn lead to the isolation of compounds namely Stigmasterol, campesterol, aervine, Betulin. The constituents isolated and characterized from the aerial parts of the plant Aerva lanata Linn can be categorized under the sterols, alkaloids and triterpenoids. From the petroleum ether (60 - 80˚) extract sterols like Stigmasterol, campesterol, from chloroform extract alkaloids like aervine were isolated and characterized by spectral data (1HNMR, 13CNMR & MASS). These compounds were isolated for the first time from the aerial parts of Aerva lanata Linn.

All these extracts and isolated compounds were subjected to analgesic activity by using two methods In acetic acid induced -writhing method petroleum ether, ethyl acetate, ethanolic extracts and isolated compounds (at doses of 200 & 400 mg/kg b.w) exhibited significant (p <0.01) analgesic activity whereas chloroform extract and isolated compound (Aervine) exhibited marked (P<0.05) analgesic activity as compared to aspirin induced control

In Tail immersion method all these extracts and isolated compounds shows significant (P < 0.01)analgesic activity at 3 hour and marked analgesic activity at 2 hour as compared to control.

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How to cite this article:

B.J. Mohan Reddy et al. ISOLATION, SPECTRAL CHARACTERIZATION AND BIOLOGICAL EVOLUTION OF PERIPHERAL ANALGESIC ACTIVITY ON AERIAL PARTS OF AERVA LANATA LINN. J Syn Natu Chem 2017;2(4):21-30.

Conflict of interest: The authors have declared that no conflict of interest exists.

Source of support: Nil


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