ISOLATION AND CYTOTOXIC POTENTIAL OF SILVER NANOSUSPENSION OF THE ROOTS OF APHANAMIXIS POLYSTACHYA

ISOLATION AND CYTOTOXIC POTENTIAL OF SILVER NANOSUSPENSION OF THE ROOTS OF APHANAMIXIS POLYSTACHYA

R. Gude ^*, A. Joshi, A. Bhandarkar and D. Mayenkar

Goa College of Pharmacy, 18^th June Road, Panaji - 403001, Goa, India.

ABSTRACT: Objective: The study was aimed to isolate the components from the methanolic extract of the root bark of Aphanamixis polystachya and to confirm their potential cytotoxicity of the methanolic extract, silver nanoparticles and silver nanosuspension of the root bark of A. polystachya. Method: Isolation from the methanolic extract of root barks of A. polystachya were performed using column chromatography technique. Cytotoxic activity on MCF-7 and MDA-MB cell lines by SRB Assay was conducted on silver nanosuspension of A. polystachya. Results: The components isolated from methanolic extract of root bark of A. Polystachya by column chromatography were confirmed as Rohituka 7, Rohituka 3, Amoorinin-3-o-α-l-Rhamnopyranosyl-(1à6)–β-D-Glucopyra-noside and 8- Methyl-7, 2׳,4׳-tri-O-Methylflavonone-5-O-α-L-Rhamnopyranosyl (1 4)-β-D- glucopyranosyl-(1 6)-β-D-glucopyranoside on the basis of IR, ¹HNMR, ¹³C NMR and MS. The prepared silver nanosuspension, tested in concentrations ranging between 500-7.81 µM showed IC₅₀ value at 0.58 µM in MCF-7 cell line, and IC₅₀ in MDA-MB cell line being greater than 1000µM. Conclusion: Column chromatography of the methanolic extract of root barks of A. polystachya led to the isolation of Rohituka 7, Rohituka 3, Amoorinin-3-O-α-L-Rhamnopyranosyl-(1à6)–β-D Glucopyranoside and (methyl-7,2׳,4׳-tri-O-methyl flavonone-5-O-α-L-rhamnopyranosyl(1à4)-β-D-glucopyranosyl-(1à6)-β-D-glucopyranoside. SRB Assay confirmed that silver nanosuspension of A. polystachya to be potentially cytotoxic. The isolated components have already proven to possess cytotoxic activity. Hence, the study suggests that silver nanosuspension exhibited better cytotoxic activity.

Aphanamixis Polystachya, Isolation, Herbal mediated Silver Nanosuspension, SRB Assay, cytotoxic activity

INTRODUCTION: Cancer is a major health problem globally, affecting 15% of the population. It is an uncontrolled growth and quick division of the abnormal cells in the body. It is projected that by 2020, the incidence of cancer levels will increase to 15 million cases, causing deaths ¹.

In the treatment of cancer, many synthetic and chemotherapeutic agents have been developed, having various side effects. Many of the plants traditionally have reported possessing antitumor activity ¹.

The medicinal plant chosen for the work is A. polystachya, which is a highly valued species for mankind and has been thoroughly investigated for its high potential medicinal value ². The design and development of herbal nanoparticles have become frontier research in the field of nanotechnology. Development of this novel drug delivery system will help in overcoming various constraints like bioavailability, solubility and stability of the herbal drug ³. The present study is to isolate the components from the methanolic extract of the root bark of the plant A. polystachya having potential cytotoxic activity. The extract was formulated into silver nanoparticles and subsequently into silver nanosuspension and assessed for cytotoxic activity by SRB method.

MATERIALS AND METHODS:

Authentication of the Plant: The root bark of A. polystachya were identified and obtained from Mangalore, Dakshina Karnataka, India. The plant part was identified and authenticated by Dr. Gopal Krishna Bhatt, Poorna Prajnya College Udupi- Karnataka, India, and Dr. Dinesh Nayak, Mangalore-Karnataka, India bearing number: GCP.Pharmacog.05/2013. The herbarium was deposited in the Department of Pharmacognosy, Goa College of Pharmacy.

Description of the Plant: A. polystachya is an evergreen medium-sized tree with a dense spreading crown and a straight cylindrical bole up to 15m in height and 1.5-1.8 m in width belonging to the family Meliaceae. In Sanskrit, it is known as Anavallabha, Ksharayogya, Lakshmi, Lakshmivana, Lohita ⁴.

Distribution: A. polystachya is native to temperate Asia, tropical China, Indian subcontinent- Peninsula, Northeast India, Bhutan, Sri Lanka, Myanmar, Thailand, Malaysia, Indonesia, Papua, New Guinea, and the Philippines. In India, it is found distributed in the Sub-Himalayan tract from the Rapti river eastwards, Sikkim up to 6000 ft., Assam, Burma, Chota Nagpur, Konkan, W. Ghats, and adjoining hill ranges from the Poona district southwards to Tinnevelly up to 3500 ft ⁵.

General Experimental Procedure: Roots, along with the bark, were collected, washed, and dried in the shade. The dried material was then powdered (500 g) and exhaustively extracted by maceration with 3 liters of methanol for 3 days. After 3 days, the methanolic layer was decanted off. The process was repeated thrice. The solvents from the total extract were distilled off using a rotary vacuum evaporator (Roteva) and concentrated to a syrupy consistency and then evaporated to dryness (150g).

Preliminary Phytochemical Screening (Qualitative Analysis): The preliminary phytochemical studies were performed to confirm the availability of different phytoconstituents present in the methanolic extract ^{2, 6}.

Synthesis of Herbal mediated Silver Nanoparticles of A. polystachya: The 10^-3 mM Silver nitrate solution was prepared. 10 ml of herbal extract was taken in 250 ml conical flask/ beakers separately, and to this 90 ml of AgNO₃ solution was added. The conical flasks were incubated at room temperature. A color change of the leaf extracts from pale yellow to dark brown was checked periodically. The brown color formation indicates that the silver nanoparticles were synthesized from the herbal extract, and they were centrifuged at 5000 rpm for 15 min in order to obtain the pellet that is used for further study. The supernatant was used for characterization ⁶.

Extraction and Isolation of Components from the Methanolic extract of the Roots of A. polystachya: The methanol soluble fraction (150g) was suspended in 1.5 L of water and extracted with petroleum ether (60:80) to remove the fatty components completely. The defatted crude extract was further partitioned with chloroform (3 × 1L) to give a chloroform soluble fraction (90g). 90g of chloroform fraction was mixed with silica gel (30g, #60-120). The sample was loaded on a column packed with 500g of silica gel (Molychem, #60 × 120) prepared in petroleum ether (60-80). The column was subjected to elution with different solvent system starting first with 100% petroleum ether followed by petroleum ether: ethyl acetate graded mixtures (95:5, 90:10, 80:20, 70:30, 50:50), 100% ethyl acetate and finally with graded mixtures of ethyl acetate: methanol (99:1, 98:2, 97:3, 96:4, 95:5).

The elutions were monitored by TLC (silica gel G, visualization by UV at 254nm, 366nm and vanillin sulphuric acid reagent heated at 110 °C). Each time 10ml of elutes were collected and identical elutes were combined (TLC monitored) and concentrated to 5ml and kept aside. The elutions carried out with petroleum ether: ethyl acetate (90:10 to 50:50) resulted in 4 fractions containing mixture of compounds and having identical R_f values (7g).

7g was mixed with flash grade silica (3g, #200-400) using mortar and pestle. This mixture was subjected for rechromatography with column (1ft in length and 2cm i.d.). The sample was loaded onto a column packed with 150g of flash grade silica (Molychem, #60 × 120). The mixture was loaded onto this column and the elutions were carried out with 100% petroleum ether, petroleum ether: ethyl acetate graded mixture (90:10, 85:50, 80:20, 75:25, 70:30, and 65:35). The elutions were monitored by TLC (silica gel G, visualization by UV at 254nm, 366nm and vanillin sulphuric acid reagent heated at 110 °C). Each time 10ml of the elutes were collected and identical elutes were combined (TLC monitored) and concentrated to 5ml and kept aside. The elutions carried out with petroleum ether: ethyl acetate (80:20) resulted in a single spot on TLC prepared in petroleum ether (80:20).

After removing the solvent, a light yellow solid was obtained which was designated as RG-APE2 (78mg). The elutions carried out with petroleum ether: ethyl acetate (65:35) resulted in a single spot on TLC prepared in petroleum ether: ethyl acetate (65:35). After removing the solvent, a light-yellow solid was obtained which was designated as RG-APE1 (74 mg). Elutions carried out with 100% ethyl acetate, ethyl acetate: methanol (99:1 and 98:2) resulted in a mixture of compounds having identical R_f values [TLC monitored, ethyl acetate: methanol (98:2)]. After removing the solvent, a light brown amorphous powder resulted (185 mg).

Purification of Compounds using Flash Chro-matography (Combi Flash Chromatograph): 185 mg of the powder was taken and mixed by triturating with flash grade silica (600 mg, #200-400) using mortar and pestle. Pre-packed silica column (Redisep RF, 1 gm) was used. All the parameters were set and monitored using peak track software.

The compounds which are UV absorbing i.e. at 254 nm and 366 nm, are only detected by flash chromatography. The elutions were carried out with ethyl acetate: methanol (98:2) and collected in a test tube. Each time, 8ml of elutes were collected and identical elutes were combined (TLC monitored, ethyl acetate: methanol 98:2), concentrated and kept aside. The test tubes from 3-18 resulted in a single spot on TLC (ethyl acetate: methanol, 98:2).

After removing the solvent, light brown flakes resulted and the compound was designated as RG-APE3 (68 mg). The test tubes from 22-44 resulted a single spot on TLC (ethyl acetate: methanol, 98:2). After removing the solvent, it resulted in a light yellow powder and the compound was designated as RG-APE4 (77 mg). The elutions carried out with other solvent systems resulted a mixture of compounds or resinous mass, which were not processed further.

Preparation of Silver Nanosuspension of A. polystachya: Silver Nanosuspension of A. polystachya (AgNS) was prepared by nano-precipitation method. The prepared A. polystachya silver nanoparticles (10 mg) were added to sodium lauryl sulphate (0.125%) in 10 ml of deionised water with continuous stirring at 500 rpm for 1 h. The solvent was allowed to evaporate to a dry nanosuspension ⁷.

Cytotoxic Assay of Nanosuspension of A. polystachya: The IC₅₀ of AgNS was determined by performing the Sulforhodamine B (SRB) Assay for cytotoxicity. The SRB assay was performed according to the described method ^{7, 8, 10} with slight modifications using concentrations of compounds ranging from 500-7.81 µM.

Absorbance was read at 540 nm on a scanning multi-well plate reader (EL × 800, BioTek Instruments Inc., Winooski, VT, USA), the percentage cell viability was calculated using excel sheet and IC₅₀ values were determined using graph pad prism. All the experiments were conducted in triplicates. The percentage cell death was calculated using the formula:

% Cell death = [(OD of control − OD of test) / (OD of control)] × 100

Characterization of Isolated Compounds:

Spectral Data:

RG-APE 1: Light yellow solid; R_f = 0.060 (solvent system CHCl₃: EtOAc 80:20); m.p.: 123 °C;

IR (KBr): 3334.92 cm^-1, 2924.25 cm^-1, 1724.36 cm^-1, 1442.75 cm^-1, 1377.17 cm^-1, 1132.21 cm^-1.

¹HNMR (CDCl₃): δ 7.5509 (d, 1H, H-1), δ 6.4724 (s, 1H, H-2), δ 2.1816 (d, 1H, H-5), δ 2.5108 (s,1H, H-6),δ 3.1835 (s, 1H, H-9), δ 5.1943 (s,1H, H-11),δ 5.8707 (t,1H, H-12), δ 5.5848 (d,1H, H-13), δ 2.0304 (t,1H, H-16), δ 3.7364 (t,1H, H-17), δ 0.9843 (s, 3H, H-18), δ 1.0638 (s,3H, H-19), δ 7.3975 (d,1H, H-21), δ 6.8561 (s, 1H, H-22),δ 7.3869 (s, 1H, H-23), δ 1.6665 (s,3H, H-28),δ 4.8607 (d, 1H, -H29), δ 5.2234 (s, 1H, H-30), δ 3.4367 (s, 1H, H-2’), δ 1.4091 (s, 1H, H-3’), δ 1.2339 (d, 1H, H-4’), δ 0.7841 (t, 1H, H-5’), δ 0.8359 (d, 3H, H-3’-Me),δ 8.1664 (s, 1H, COOH), δ 2.0470 (s, 3H, CH₃COO).

¹³CNMR (CDCl₃): δ 153.08 (C-1), δ 122.42 (C-2), δ 169.0 (C-3), δ 81.93 (C-4), δ 74.06 (C-5), δ 31.25 (C-6), δ 174.83 (C-7), δ 138.50 (C-8), δ 50.70 (C-9), δ 39.55 (C-10), δ 74.06 (C-11), δ 80.37 (C-12), δ 61.20 (C-13), δ 81.90 (C-14),δ 72.30 (C-15), δ 39.34 (C-16), δ 39.14 (C-17), δ 11.53 (C-18), δ 28.67 (C-19), δ 123.05 (C-20) δ 110 (C-22), δ 143.0 (C-23), δ 28.90 (C-28), δ 74.06 (C-29), δ 119.30 (C-30), δ 174.83 (C-1’), δ 74.06 (C-2′), δ 39.01.25 (C-3′), δ 22.06 (C-4′), δ 11.39 (C-5′), δ 15.30 (C-3′-Me), δ 150.08 (C-HCOO), δ 159.08 (C-CH₃COO), δ 20.7 (C-CH₃COO).

Mass Spectra: (LC-MS): C₃₅H₄₄O_13, mol. wt: 672; LC-MS (m/z): 672.16 (M+)⁺. The other peaks appeared at 672.27, 643.28, 511.20, 451.18, 433.17, 339.12, 269.12, 165.07, 152.08. From the m.p, IR, ¹HNMR, ¹³CNMR, and MS, compound RG-APE1 was designated as Rohituka 7.

FIG. 1: CHEMICAL STRUCTURE OF RG-APE1 (ROHITUKA 7)

RG-APE2: Light yellow solid; R_f value: 0.065 (solvent system CHCl₃: EtOAc 80:20); m.p.: 122.4 °C; positive response for Liebermann- Burchard test for triterpenoids.

IR (KBr): 3389.10 cm^-1, 2931.25 cm^-1, 1741.57 cm^-1, 1448.90 cm^-1, 1379.09 cm^-1, 1031.08 cm^-1.

¹HNMR (CDCl₃): δ 3.7364 (t, 1H, H-1), δ 2.9528 (d, 1H, H-2), δ 2.3029 (d 1H, H-5), δ 2.8218 (m, 1H, H-6), δ 3.1835 (s, 1H, H-9), δ 4.8607 (s, 1H, H-11), δ 5.8708(t,1H, H-12), δ 2.841, 2.3360 (t,1H, H-16), δ 3.7277 (t,1H, H-17), δ 0.8596 (s,1H, H-18), δ 1.1986 (s, 3H, H-19), δ 7.3975 (d,1H, H-21), δ 6.4724 (s,1H, H-22), δ 7.5509 (s, 1H, H-23),δ 1.6665 (s, 1H, H-28), δ 4.9521, 3.7227 (t,1H, H-29),δ 6.8561(s, 1H, H-30), δ 3.4367 (s, 1H, H-2’), δ 1.7908 (d, 1H, H-3’), δ 1.1606, 0.9428 (t, 1H, H-4’), δ 0.6985 (s, 1H, H-5’), δ 0.8934 (t, 3H, H-3’-Me).

¹³CNMR (CDCl₃): δ 78.26 (C-1), δ 28.98 (C-2), δ 167.06 (C-3), δ 80.37 (C-4), δ 39.34 (C-5), δ 31.25 (C-6), δ 169.86 (C-7), δ 123.05 (C-8), δ 54.70 (C-9), δ 50.70 (C-10), δ 74.06 (C-11),δ 78.26 (C-12), δ 39.55 (C-13), δ 81.93 (C-14),δ 204.08 (C-15), δ 39.14 (C-16), δ 28.67 (C-17), δ 15.30 (C-19), δ 119.32 (C-20), δ 139.55 (C-21) δ 111.22 (C-22), δ 143.20 (C-23), δ 22.06 (C-28), δ 74.06 (C-29), δ 122.42 (C-30), δ 174.83 (C-1’), δ 74.06 (C-2′), δ 31.25 (C-3′), δ 20.70 (C-4′), δ 11.39 (C-5′), δ 11.53 (C-3′-Me).

Mass Spectra (LC-MS): C₃₂H₄₀O₁₁, Mol. wt: 600, (M+)⁺. The other peaks appeared at 599.36, 557.35, 497.33, 479.32, 407.26, 189.07, 152.06. From the m.p, IR, ¹HNMR, ¹³CNMR and Mass spectra, com-pound RG-APE2 was designated as Rohituka 3.

FIG. 2: CHEMICAL STRUCTURE OF RG-APE2 (ROHITUKA 3)

RG-APE3: Light brown flakes; R_f value: 0.371 (solvent system 100% EtOAc); m.p.: 189 °C; positive response for Molisch’s test for carbohydrates and belongs to the limonoid class.

IR (KBr): 3456.11 cm^-1, 1745.0 cm^-1, 1641.22 cm^-1, 820.39 cm^-1, 860.37 cm^-1, 1244.4 cm^-1, 893.29 cm^-1, 820.39 cm^-1

¹HNMR (DMSO): δ 0.96 - δ 1.05 (s, 12H, H-18, H-19,H-24, H-25), δ 1.06, 1.33, 1.40,1.17 (s,4H, H-11,12), δ 2.26(d,1H, H-5), δ 2.27 (s,2H, H-6),δ 2.28 (s,1H, H-9), δ 3.39 (s,1H, H-3),δ 3.78 (s,3H, H-27), δ 4.88 (s,1H, H-15),δ 4.91, δ 5.09 (each s,2H H-26), δ 5.63 (s,1H, H-17), δ 5.68 (s,1H, H-1),δ 5.86 (s,1H, H-2,), δ 6.43-δ 7.59 (m,3H, H-21,22,23 of furan), δ 1.27 (s, 3H, H-6′′), δ 3.80- δ 4.49(m, 4H, H-2′,3′,4′5′), δ 4.52 (s,1H, H-1′′), δ 4.54- δ 4.68(m,4H, H-2′′,3′′,4′′,5′′),δ 4.70-4.87 (m,6H, H-2′,3′,4′,2′′,3′′,4′′-OH), δ 4.93 (s,2H, H-6′), δ 5.13 (s,1H, H-1′).

¹³CNMR (DMSO): δ 136.14 (C-1), δ 124.59 (C-2), δ 105.44 (C-3), δ 37.51 (C-4), δ 39.93 (C-5), δ 39.51 (C-6), δ 172.93 (C-7), δ 149.50 (C-8), δ 40.14 (C-9), δ 28.96 (C-10),δ 27.21 (C-11), δ 28.96 (C-12), δ 39.72 (C-13),δ 78.50 (C-14),δ 59.64 (C-15), δ 170.14 (C-16), δ 86.50 (C-17), δ 11.41 (C-18), δ 14.00 (C-19), δ 120.14 (C-20) δ 101.59 (C-21), δ 140.16 (C-22) δ 142.93 (C-23), δ 20.66 (C-24), δ 20.41 (C-25), δ 114.14 (C-26) δ 49.85 (C-27), δ 110.16 (C-1′), δ 90.50 (C-2′′), δ 70.14 (C-3′), δ 79.03 (C-4′), δ 79.16 (C-5′) δ 39.30 (C-6′), δ 112.12 (C-1′′) ,δ78.83 (C-2′) δ 87.59 (C-3′′), δ 88.03 (C-4′′) δ 68.16 (C-5′′), δ 15.44 (C-6′′).

Mass Spectra (ESI-MS): C₃₉H₅₄O₁₆, mol.wt.: 778.84, 778.1 (M+)⁺. The other peaks appeared at 712.4, 677, 623.3, 560.4, 482, 390.7, 278.8, 144.9.

FIG. 3: CHEMICAL STRUCTURE OF COMPOUND RG-APE3(Amoorinin-3-O-α-L-Rhamnopyranosyl-(1,6) –β-D- Glucopyranoside)

From the m.p., IR, ¹HNMR, ¹³CNMR, and MS, compound RG-APE3 was designated as Amoorinin-3-O-α-L-Rhamnopyranosyl–(1à6) –β-D-Glucopyranoside.

RG-APE4: Yellow Solid; R_f value: 0.06 (solvent system CHCl₃: EtOAc 60:40); m.p.: 212 °C; positive test for flavanone glycoside.

IR (KBr): 3447.18 cm^-1, 2963.12 cm^-1, 2877.24 cm^-1, 1644.29 cm^-1, 1461.18 cm^-1, 1377.10 cm^-1, 1234.4 cm^-1, 1147.6 cm^-1, 800.4 cm^-1, 828.41 cm^-1.

¹HNMR (DMSO): δ1.24 (s, 3H, H-6′′′′- Me), δ 2.13 (s, 3H, H-8 Me), δ 2.53 (s,1H, H-5′′), δ 3.39 (s,1H, H-3′′), δ 3.58 (s,2H, H-6′′), δ 3.69 (s,2H, H-6′′′), δ 3.72 (s,1H, H-2′′′′), δ 3.77 (s,2H, H-3), δ 3.82-3.87 (m,9H, H-2′′,3′′,4′′,2′′′,3′′′,6′′′,2′′′′,3′′′′4′′′′-OH), δ 3.80, 3.93, 4.00 (s, 9H, H-2′, 4′, 7-3xOMe) δ 6.04 (d, 1H, H-1′′), δ 6.05 (d, 1H, H-1′′′), δ 5.07 (d,1H, H-1′′′′), δ 5.38 (m,1H, H-2), δ 6.63 (s,1H, H-6), δ 6.95 (s,1H, H-3′), δ 7.30 (d,1H, H-5′), δ 7.32 (d,1H, H-6′), δ 4.02 (s,1H, H-2′′), δ 4.10 (s,1H, H-4′′), δ 4.20 (s,1H, H-2′′′), δ 4.54 (s,1H, H-3′′′), δ 4.55 (s,1H, H-3′′′′), δ4.56 (s,1H, H-4′′′), δ 4.58 (d, 1H, H-4′′′′), δ 5.04 (d, 1H, H-5′′′), δ5.07 (m, 1H, H-5′′′′).

¹³CNMR (DMSO): δ 83.03 (C-2), δ 40.15 (C-3), δ 195.15 (C-4), δ 102.89 (C-4a), δ 164.86 (C-5) δ 130.13 (C-6), δ 168.37 (C-7), δ 115.37 (C-8) δ 165.67 (C-8a), δ 11.68 (C-8-Me) δ 129.13 (C-1′),δ 155.67 (C-2′), δ 114.86(C-3′),δ 159.03 (C-4′) δ 116.52 (C-5′), δ119.03 (C-6′), δ 55.67, 56.62, 50.15 (C-2′,4′,7-OCH₃ x3), δ 103.03 (C-1′′), δ 78.37 (C-2′′), δ 39.52 (C-3′′), δ 31.94 (C-4′′), δ 81.30 (C-5′′), δ 38.89 (C-6′′), δ 94.71 (C-1′′′), δ 39.94 (C-2′′′), δ 79.03 (C-3′′′), δ 31.30 (C-4′′′), δ 28.71 (C-5′′′), δ 25.02 (C-6′′′), δ 99.94 (C-1′′′′), δ 39.73 (C-2′′′′), δ 39.31 (C-3′′′′), δ 29.03 (C-4′′′′), δ 28.62 (C-5′′′′), δ 18.79 (C-6′′′′).

FIG. 4: CHEMICAL STRUCTURE OF COMPOUND RG-APE4 – (methyl-7,2׳,4׳-tri-O-methyl flavonone-5-O-α-L-rhamnopyranosyl (1à4)-β-D-glucopyranosyl-(1à 6)-β-D-glucopyranoside)

Mass Spectra (ESI-MS): C₃₇H₅₀O₂₀, mol. wt.: 814.78, 816.1 (M+, 2H) ⁺. The other peaks are observed at 751.1, 572.4, 332.7, 159.8, 145.9, 128, 113.9. From the m.p., IR, ¹HNMR, ¹³CNMR and MS, compound RG-APE4 was designated as 8- methyl- 7, 2׳, 4׳- tri- O- methylflavonone-5-O-α-L-rhamno-pyranosyl (1 4)-β-D-glucopyranosyl-(1 6)-β-D-glucopyranoside.

RESULTS: Extensive column chromatography isolated four compounds, Rohitika 7 Fig. 1, Rohituka 3 Fig. 2, Amoorinin-3-O-α-L-Rhamno-pyranosyl-(1à6) –β-D- Glucopyranoside Fig. 3 and Methyl-7,2׳,4׳-tri-O-methyl flavonone-5-O-α-L-rhamnopyranosyl (1à4)- β- D-glucopyranosyl-(1à6)-β-D-glucopyranoside Fig. 4. The structures of the isolated compounds were established by interpretation of their spectroscopic data, by IR, ¹HNMR, ¹³CNMR and MS with those published in the reference papers.

The formulated A. polystachya nanosuspension was tested in concentration ranges between 500-7.81 µM on MCF-7 and MDA-MB cell lines. It was found that the IC₅₀ value obtained was 0.58 µM in MCF-7 cell line, and the IC₅₀ in MDA-MB is greater than 1000.

DISCUSSION: In this study, an attempt was made to isolate the components from the methanolic extract of the root bark extract of A. polystachya. 

The compound RG-APE1 showed an m.p. at 123.0 °C and positive response for Liebermann- Burchard test for triterpenoids. The IR spectrum showed a strong absorption at 3334.92 cm^-1, indicating the presence of the hydroxyl group. The band at 2924.25 cm^-1 indicated C-H stretching in CH₃. The prominent peak at 1724.36 cm^-1 shows carbonyl stretching of ester. The ¹HNMR displayed exhibited a singlet at δ 5.2234, indicating methyl protons at C-30. The doublet at δ 7.3975 (C-21) and two singlets at δ 6.8561 (C-22) and δ 7.3869 (C-23) indicated the presence of protons in the furan moiety. The peak at δ 8.1664 indicated the presence of the carboxylic group. A singlet at δ 2.0470 indicated three methyl protons of CH₃COO. A singlet at δ 3.4367 indicated the presence of proton at C-2’. The value at δ 1.4091 appeared as a singlet and δ 1.2339 a doublet indicating two protons at c-3,’ and C-4‘.δ0.8359 appeared as a doublet indicating three protons at C-3’-Me. The ¹³CNMR spectrum exhibited values at δ 174.83 (C-7), δ 169.0 (C-3), indicating the presence of two ester groups. δ 150.08 indicated the carbon for COOH. The value at δ 159.08 indicated the presence of carbon for CH₃COO.

The LC-MS spectrum displayed the molecular ion peak at m/z 672 correspondings to the molecular formula C₃₅H₄₄O₁₃. From the above evidence, the compound RG-APE1was designated as Rohituka 7.

The compound RG-APE2 showed a m. p. at 122.4 °C. It showed a positive response for Liebermann- Burchard test for triterpenoids. The IR spectrum showed a strong absorption at 3389.10 cm^-1, indicating the presence of the hydroxyl group. The band at 2931.25 cm^-1 indicated C-H stretching in CH₃. The prominent peak at 1741.57 cm^-1 shows carbonyl stretching of ester. The ¹HNMR of this compound exhibited a singlet at δ 6.8561indicating methyl protons at C-30. The value at doublet δ 7.3975, singlets δ6.4724 and δ 7.5509 indicated the presence of furan moiety. Here it is clearly noted that the value of COOH and CH₃COO are absent when compared to Rohituka 7. A singlet at δ 3.4367 and δ 1.7908 indicated the presence of protons at C-2’ and C-3’ and d 1.1606 at C-4’. The proton at C-5 appeared at 0.6985 as a single. A triplet appeared at δ 0.8934, indicating 3 methyl groups at 3’- Me. The ¹³CNMR spectrum exhibited a peak at δ 167.06 (C-3), δ 169.0 (C-7), indicating the presence of two ester groups. The LC-MS spectrum displayed the molecular ion peak at m/z 600 correspondings to the molecular formula C₃₂H₄₀O₁₁. From the above evidence, the compound RG-APE2was designated as Rohituka 3.

The compound RG-APE3 showed a m. p. at 189 °C and a positive response to Molisch’s test for carbohydrates and belong to the limonoid class. The IR spectra showed a peak at 3456.11 cm^-1, indicating the presence of the hydroxyl group. The peak at 1745.0 cm^-1 corresponds to the carbonyl group of the ester. The peaks at 1641.22 cm^-1 and 820.39 cm^-1 indicated a trisubstituted double bond. The peak at 860.37 cm^-1 is due to the presence of furan ring. The peak at 1244.4 cm^-1 corresponds to the epoxide moiety. The peak at 820.29 cm^-1 is due to the glycoside moiety.

In the proton NMR spectra, the singlets at δ 0.96, δ 0.99,δ 1.09,δ 1.05 corresponds to 12 hydrogens of 4 methyl groups at C-18, 19, 24, 25. The singlet at δ 1.27 corresponds to the methyl protons of the rhamnose at C-6′′. The value at δ 5.13 corresponds to 1 hydrogen of C-1′ of a glucose molecule. The multiplets at δ 6.43- δ 7.59 is due to three hydrogens of the furan moiety. The singlet at δ 4.91, δ 5.09, corresponds to methylene protons at C-26.

The ¹³CNMR spectra showed signals at δ 172.93 (C-7), δ 170.14 (C-16) suggesting the presence of two ester groups. The signals at δ 142.93 (C-23), δ 140.16 (C-22), δ 101.59 (C-21), δ 120.14 (C-20) are characteristic of furan ring. The LC-MS spectrum indicated the molecular ion peak at 778.1 [M+] corresponding to the molecular formula C₃₉H₅₄O₁₆.

From the above evidences the compound RG-APE3 was designated as Amoorinin-3-O-α-L-rhamno-pyranosyl – (1à6) –β-D-glucopyranoside.

The compound RG-APE4 showed m.p. at 212 °C and a positive test for flavanone glycoside. The IR spectra showed peaks at 3447.18 cm^-1, indicating the presence of hydroxyl group. The peak at 2963.12 cm^-1 corresponds to C-H stretching of CH₃. The peak at 2877.24 cm^-1 corresponds to the C-H stretching of in CH₃ of methoxy group. The peaks at 1644.29 cm^-1, 1461.18 cm^-1, 1377.10 cm^-1, 1234.4 cm^-1, 1147.6 cm^-1, 800.4 cm^-1 is due to C-H stretching in flavonone nucleus. The peak at 828.40 cm^-1 is due to a glycoside. In the proton NMR spectra, the singlet at δ 1.24 is due to methyl protons of the third glucose moiety at C-6′′′′. The doublet at δ 5.07 corresponds to the protons of C-1′′′′of glucose, the singlet at each δ 3.80, 3.93, and 4.00 is due to the protons of the 3 methoxy group in the structure. The multiplets at δ 3.82-3.87 account for hydroxyl protons. The singlet at δ 2.13 accounts for the methyl protons at C-8.

The ¹³CNMR shows signals at δ 55.67, 56.62, and 50.15 representing the 3 methoxy groups. The value at δ 195.15 indicates one carbonyl group. The value at δ 11.68 was indicative of one methyl group at C-8. The LC-MS spectrum indicated the molecular ion peak at 816.11 [M+] corresponding to the molecular formula C₃₇H₅₀O₂₀. From the above evidences the compound RG-APE4was designated as 8- methyl-7, 2׳, 4׳-tri-O-methyl-flavonone-5-O-α-L- rhamnopyranosyl (1à4)-β-D- glucopyranosyl- (1à6) -β- D-glucopyranoside.

The isolated compounds from the methanolic extract of root bark of A. polystachya have been reported to have potential anti-cancer activity on MCF-7 and MDA-MB cell lines ⁸. The cytotoxic studies were performed on Silver Nanosuspension of A. polystachya. It was seen that the formulation showed potent cytotoxic activity. This activity can be attributed to the isolated components which have been already reported to possess potential cytotoxic effects against human cell lines ⁹. It can thus be concluded that the silver nanosuspension of A. polystachya can be suggested for breast cancer activity, which is eco-friendly, cost-effective, and stable.

CONCLUSION: The investigation justifies that the isolated components from the methanolic extract of the root bark of A. polystachya, RG-APE1, RG-APE2, RG-APE3, and RG-APE4 have been proven to have cytotoxic activity ⁸. Based on these preliminary investigations, we formulated the root bark extract of A. polystachya into silver nano-particles and subsequently into silver nanosuspension which exhibited better cytotoxic activity than the root bark extract of the plant. The use of AgNP has emerged as a novel approach in cancer therapy. These studies suggest that formulating into AgNS can be further used in drug targeting the anti-cancer cells and create an impact on the human health

ACKNOWLEDGEMENT: The authors wish to thank authorities of the Directorate of Technical Education, Govt. of Goa and Principal, Goa College of Pharmacy for the facilities provided to carry out the research work. The authors also wish to thank Panjab University, Chandigarh-India, for carrying out the NMR and Mass Spectrometry studies on the methanolic extract of root bark of A. polystachya and Manipal College of Pharmaceutical Sciences, Manipal- India for carrying out the cytotoxic study on the nanosuspension of A. polystachya.

CONFLICTS OF INTEREST: The authors confirm that this article content has no conflict of interest.

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    How to cite this article:

    Gude R, Joshi A, Bhandarkar A and Mayenkar D: Isolation and cytotoxic potential of silver nanosuspension of the roots of Aphanamixis polystachya. Int J Pharm Sci & Res 2020; 11(11): 5766-74. doi: 10.13040/IJPSR.0975-8232.11(11).5766-74.

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