Lemuni Leaves
Vitex negundo L.
Verbenaceae
Figure 1 : Vitex negundo. (a) Whole plant; (b) leaves; (c) abaxial leaves; (d) flowers. (Photos courtesy of FRIM, 2012 & 2016)
DEFINITION
Lemuni leaves consist of the powder of dried leaves of Vitex negundo L. (Verbenaceae).
SYNONYM
Vitex incisa Lamk, Vitex paniculata Lamk, Vitex leucoxylon Blanco [ 1 ].
VERNACULAR NAMES
Five-leaved chaste tree, horseshoe vitex (English); Lagundi, lemuning, lenggundi (Malay); Nirgundi, nocchi (Indian); Huang jing (Chinese) [ 1 , 2 , 3 ].
CHARACTER
IDENTIFICATION
Plant Morphology
V. negundo is a deciduous shrub or small tree up to 8 m tall. Bark surface slightly rough, peeling off in papery flakes, pale reddish-brown. Leaves narrowly elliptical to ovate-lanceolate, minutely puberulous or glabrous above, densely tomentose or puberulent below, leaflets 3–5, median leaflet 5–15 cm x 1–4 cm, with 2–12(–18) pairs of lateral veins, median leaflet on a 1–2.5 cm long petiolule, lateral ones sometimes subsessile; cymes arranged in panicles which are terminal and axillary in the upper leaf axils. Flower calyx 1–2 mm long, shortly 5-toothed, corolla blue-violet, villous inside. Fruit globose to broadly ovoid, 3–6 mm long, purple or black when mature [ 1 ].
Microscopy
Powdered material consists of adaxial epidermis cells with straight to wavy anticlinal wall; fragment of annular vessel and fibres; fragment of trichomes mostly simple multiseriate, glandular and stellate trichome and also abundant of oil globules [ 4 ].
Figure 2 : Microscopic characters of Vitex negundo leaves powder of 0.355 mm size. (a) Adaxial epidermis cells (magnification 20x); (b) oil globules (magnification 20x); (c) fibre (magnification 20x); (d) annular vessel (magnification 40x); (e) simple, muticellular trichome (magnification 40x); (f) stellate trichome (magnification 10x). [Scale bars: a = 20 µm; b = 20 µm; c = 20 µm; d = 10 µm; e = 10 µm; f = 50 µm]
Colour Tests
Observed colour of solution after treatment with various reagents:
| NaOH (5%) | Yellow |
| KOH (5%) | Dark Yellow |
Thin Layer Chromatography (TLC)
Figure 3 : TLC chromatogram of agnuside (S) and ethanol extract of1 Vitex negundo dried leaves powder (L) observed under (a) visible light after derivatisation, (b) UV at 254 nm before derivatisation and (c) UV at 366 nm after derivatization.
| Test Solutions | Weigh about 0.5 g of V. negundo dried leaf powder of 0.355 mm size in a 14 mL screw-capped vial. Add 5 mL of ethanol and sonicate for 15 minutes at ambient temperature. Filter the solution through filter paper. Evaporate to dryness and reconstitute with 2 mL methanol. Use the solution as test solution. |
| Standard solution | Dissolve agnuside standard [CAS no.: 11027-63-7] in methanol to produce 1.0 mg/mL solution. |
| Stationary Phase | HPTLC silica gel pre-coated plate 60 F254, 10 x 10 cm |
| Mobile phase | Ethyl acetate : acetic acid : formic acid : water; (100 : 11 : 11 : 26) (v/v/v/v) |
| Application |
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| Development distance | 7 cm |
| Drying | Drying method |
| Detection |
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High Performance Liquid Chromatography (HPLC)
| Test solution | Weigh about 0.5 g of V. negundo dried leaf powder of 0.355 mm size in a 14 mL screw-capped vial. Add 5 mL of ethanol and sonicate for 15 minutes at ambient temperature. Filter the solution and evaporate to dryness. Reconstitute with 6 mL methanol and use the solution as test solution. | ||||||||||||||||||
| Standard solution | Dissolve agnuside standard [CAS no.: 11027-63-7] in methanol to produce 1.0 mg/mL solution. | ||||||||||||||||||
| Chromatographic system |
Detector: PDA 258 nm Column: C18 (5 µm, 4.6 mm i.d. x 250 mm) (Xbridge, Waters, if necessary) Column oven temperature: Ambient Flow rate: 1.0 mL/min Injection volume: 10 µL for agnuside; 3 µL for test solution |
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| Mobile Phase (gradient mode) |
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| System suitability requirement |
Perform at least five replicate injections of agnuside (1.0 mg/mL) The requirements of the system suitability parameters are as follow:
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| Acceptance criteria |
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(a)
(b)
Figure 4 : Whole HPLC chromatogram of (a) agnuside standard solution (1.0 mg/mL) at tr= 14.770 min and (b) ethanol extract of Vitex negundo dried leaves powder showing peak corresponding to agnuside standard solution at tr= 14.789 min.
(a)
(b)
Figure 5 : HPLC chromatogram highlighting the elution region of agnuside in (a) agnuside standard solution (1.0 mg/mL) at tr= 14.770 min and (b) ethanol extract of Vitex negundo dried leaves powder showing peak corresponding to agnuside standard solution at tr= 14.789 min.
Figure 6 : UV spectrum of agnuside standard solution (1.0 mg/mL) and ethanol extract of Vitex negundo dried leaves powder.
PURITY TESTS
The purity tests are based on V. negundo dried plant part powder of 0.355 mm particle size.
| Foreign Matter |
| Not more than 2% |
| Ash Contents | |
| Total ash | Not more than 7% |
| Acid-insoluble ash | Not more than 1% |
| Loss on Drying |
| Not more than 13% |
| Extractive Values | |
| Water-soluble extracts | |
| Hot method | Not less than 30% |
| Cold method | Not less than 23% |
| Ethanol-soluble extracts | |
| Hot method | Not less than 22% |
| Cold method | Not less than 13% |
SAFETY TESTS
| Heavy Metals | |
| Arsenic | Not more than 5.0 mg/kg |
| Mercury | Not more than 0.5 mg/kg |
| Lead | Not more than 10.0 mg/kg |
| Cadmium | Not more than 0.3 mg/kg |
| Microbial Limits | |
| Total bacterial count | Not more than 105 cfu/g |
| Total yeast and mould count | Not more than 104 cfu/g |
| Bile-tolerant gram negative | Not more than 104 cfu/g |
| Specific Pathogens | |
| Salmonella spp. | Absent in 25 g |
| Escherichia coli | Absent in 1 g |
| Staphylococcus aureus | Absent in 1 g |
| Pseudomonas aeruginosa | Absent in 1 g |
CHEMICAL CONSTITUENTS
Methanol extract of the leaves was found to contain monoterpene (e.g. ascaridole epoxide), iridoid (e.g. negundoside, agnuside), sesquiterpenes (e.g. caryophyllene, ledol and aromadendrene oxideI), diterpene (e.g. phytol), flavonoids (e.g. luteolin, pentamethoxy flavones and vitexin), carboxylic acids (e.g. benzoic acid, 4-hydroxybenzoic acid, 3-hydroxybenzoic acid and 3,4-dihydroxybenzoic acid), fatty acids (e.g. 6,9,12,15-docosatetraenoic acid, methyl ester and linolenic acid), carboxylic acid ester (e.g. ethyl hexanoate), steroid (e.g. ethyl iso-allocholate), sugars (e.g. 6-O-α-D-galactopyranosylD-glucose and d-mannose) and others (e.g. 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one, 1,1,3-triethoxypropane, 1,1-diethoxy-3-methylbutane, 2,3-dihydrothiophene 1,1-dioxide, (2E)-3-propyl-2,4-pentadien-1-ol, ethyl (4E)-2-nitro-4,9-decadienoate, methyl octadeca-10,13-dienoate, methyl 4-decynoate, 2-[(9Z)-9-octadecen-1-yloxy]ethanol, 2-cis,cis-9,12-octadecadienyloxy ethanol, 7α-isopropenyl-4,5-dimethyloctahydroinden-4-yl)methanol, 2-methyl-4-(2,6,6-trimethylcyclohex-1-enyl)but-2-en-1-ol, 12-bromo-13-hydroxy-2,5,9,13-tetramethyltetradeca-4,8-dienoic acid, methyl ester, 1,4-dimethyl-7-(1-methylethyl) azulene and vitamin E) [ 5 , 6 , 7 ].
Ethanol extract of the leaves was found to contain flavonoids (e.g. kaempferol, myricetin, quercetion, quercetagetin, isorhamnetin and luteolin), alkaloids, tannins, saponins, sterols, phenolic compounds, glycosides, reducing sugars, proteins and amino acids [ 9 , 10 ].
Chloroform extract of the leaves was found to contain flavone such as vitexicarpin [ 11 ].
Essential oils of the leaves was found to contain flavonoids (e.g. 5-hydroxy 3, 6, 7, 3’, 4’-pentamethoxy flavone and 3, 5-dihydroxy-6, 7, 3’, 4’-tetramethoxyflavonal), monoterpene hydrocarbons (e.g. sabinene, terpinen-4-ol, p-cymene, cis-ocimene, citronellal, α-thujene, α-pinene, camphene and linalool), sesquiterpene hydrocarbons (e.g. viridiflorol, β-caryophyllene, β-curcumene, α-guaiene, δ-guaiene, guaia-3,7-diene, valencene, (E)-nerolidol, globulol, epi- α-cadinol, α-muurolol, α-bisabolol acetate, α-copaene and β-elemene), oxygenated sesquiterpenes (e.g. humulene epoxide I, humulene epoxide II and caryophyllene epoxide), diterpenoids (e.g. epi-laurenene and abietadiene), fatty acids (e.g. palmitic acid, stearic acid and behenic acid), glycosides (e.g. negundoside, nishinadaside), carboxylic acid esters (e.g. ethyl-9-hexadecenoate) and other hydroxybenzoylmussaenosidic acid derivatives [ 12 , 13 , 14 , 15 ].
MEDICINAL USES
Uses described in folk medicine, not supported by experimental or clinical data
Traditionally, the leaves are regarded as a tonic and febrifuge. A decoction of the leaves is used for head-ache, catarrh, rheumatism and beri-beri [ 16 ].
Biological and pharmacological activities supported by experimental data
Antimicrobial activity
Essential oil of V. negundo leaves (1.0 v/v) inhibited the growth of Staphylococcus aureus with inhibition zone of 24 mm, Klebsiella pneumonia (12 mm), Bacillus subtilis (12 mm), Micrococcus luteus (18 mm) compared to ciprofloxacin (11 – 26 mm). The oil also inhibited the growth of Candida albicans (13 mm)compared to chloramphenicol (11 – 14 mm) using agar diffusion assay [ 13 ].
Benzene extract of V. negundo leaves (20 mg/mL) inhibited the growth of S. aureus with inhibition zone of 19.33 mm and minimum inhibitory concentration (MIC) of 2.5 mg/mL, Bacillus subtilis (17.33 mm, MIC = 2.5 mg/mL), E. coli (18.33 mm, MIC = 1.25 mg/mL), P. aeruginosa (17 mm, MIC = 5.0 mg/mL) and Proteus vulgaris (17 mm, MIC = 5.0 mg/mL) compared to tetracycline (22 – 24.33 mm) using agar well diffusion assay [ 15 ].
Water-ethanol (50 : 50) extract of V. negundo leaves (20 mg/mL) inhibited the growth of S. aureus with inhibition zone of 18.66 mm and minimum inhibitory concentration (MIC) of 2.5 mg/mL, Bacillus subtilis (19 mm, MIC = 5.0 mg/mL), E. coli (19 mm, MIC = 2.5 mg/mL), P. aeruginosa (18.33 mm, MIC = 2.5 mg/mL) and Proteus vulgaris (17.66 mm, MIC = 2.5 mg/mL) compared to tetracycline (22 – 24.33 mm) using agar well diffusion assay [ 15 ].
Methanol extract of V. negundo leaves (200 µg/disc) inhibited the growth of Bacillus cereus with inhibition zone of 10 mm, Pseudomonas aeruginosa (10 mm)and Salmonella typhi (10 mm)compared to kanamycin (10 – 15 mm) using disc diffusion assay [ 17 ].
Petroleum-ether and carbon tetrachloride fraction of V. negundo ethanol extractleaves (200 mg/mL) inhibited the growth of Bacillus megaterium, Bacillus subtilis, Salmonella typhi and Vibrio mimicus with inhibition zone ranging from 10 to 18 mm compared to kanamycin (8 – 19 mm) using disc diffusion assay [ 17 ].
Ethanol extract of V. negundo leaves (50 mg/100 μL) inhibited the growth of Xanthomonas axonopodis with inhibition zone of 20.5 mm and Pseudomonas solanacearum (21.9 mm) compared to streptomycin (22.1 mm and 24.0 mm respectively) using agar well diffusion assay [ 18 ].
Ethyl acetate extract of V. negundo leaves (50 mg/100 μL) inhibited the growth of X. axonopodis (24.2 mm) and P. solanacearum (21.2 mm) compared to streptomycin (22.1 mm and 24.0 mm respectively) using agar well diffusion assay [ 18 ].
Penta methoxy flavone isolated from methanol extract of V. negundo leaves (500 mg/mL) inhibited the growth of Streptococcus (15 mm), Klebsiella (12 mm), Proteus mirabilis (10 mm)and E. coli (11 mm)compared topenicillin, gentamycin and streptomycin (8 – 33 mm) using disc diffusion assay [ 19 ].
Benzene extract of V. negundo leaves (20 mg/mL) inhibited the growth of Aspergillus niger with inhibition zone of 19.33 mm and minimum inhibitory concentration of (MIC) 5.0 mg/mL, Candida albicans (20.33 mm, MIC = 10 mg/mL) and Aspergillus flavon (19.33 mm, MIC = 10 mg/mL) compared to tetracycline (27 – 29 mm) using agar well diffusion assay [ 15 ].
Water: ethanol (50 : 50) extract of V. negundo leaves (20 mg/mL) inhibited the growth of A. niger with inhibition zone of 18.33 mm and minimum inhibitory concentration of (MIC) 5.0 mg/mL, C. albicans (17.33 mm, MIC = 10 mg/mL) and A. flavon (18.33 mm, MIC = 5.0 mg/mL) compared to tetracycline (27 – 29 mm) using agar well diffusion assay [ 15 ].
Antiarthritic activity
Aqueous extract of V. negundo leaves (1000 µg/mL) showed antiarthritic activity to protein denaturation (fresh egg albumin) with inhibition rate 88.40% compared to diclofenac sodium (inhibition rate: 80.24%) [ 20 ].
Antifilarial activity
Crude ethyl acetate extract of V. negundo leaves (0.03 – 1.00 mg/mL) showed antifilarial activity against Setaria cervi in dose-dependent manner with inhibition rate (14.2 to 98.7%) compared to untreated control using MTT reduction assay [ 21 ].
Antidiabetic activity
Aqueous extract of V. negundo leaves (500 mg/kg) was administred orally to alloxan-induced diabetic Wistar albino rats once daily for 6 weeks. The extract showed significant (p < 0.01) reduction in blood glucose level (102.2 ± 1.3 mg/dL) compared to glibenclamide treated group (83.2 ± 1.3 mg/dL) [ 22 ].
Ethanol extract of V. negundo leaves (500 mg/kg) was administred orally to alloxan-induced diabetic Wistar albino rats once daily for 6 weeks. The extract showed significant (p < 0.05) reduction in blood glucose level (116.2 ± 1.3 mg/dL) compared to glibenclamide treated group (83.2 ± 1.3 mg/dL) [ 22 ].
Anti-inflammatory activity
Methanol extract of V. negundo leaves (5 µg/ear) was applied topically to mouse ear 40 minutes before 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced edema. The extract inhibited the edema (74%) compared to indomethacin (97%) [ 5 ].
Ethanol extract of V. negundo leaves (500 mg/kg) was injected peritoneally to mice 30 minutes before induction of paw edema using carrageenan. After five hours, the extract significantly (p < 0.01) inhibited paw edema thickness (78.1%) compared to diclofenac (70%) [ 23 ].
Ethanol extract of V. negundo leaves (100 and 200 mg/kg) was injected subcutaneously into the right hind paw of adult Wistar albino rats (180 – 230 g) one hour before induction of paw edema using carrageenan. After five hours, the extract inhibited paw edema thickness (100 mg/kg: 48.13%; 200 mg/kg: 57.18%) compared to indomethacin (61.16%) [ 24 ].
Antioxidant activity
Aqueous extract of V. negundo leaves (100 – 1400 μg/mL) showed antioxidant activity with 2,2-azinobis(3-ethyl benzothiazoline-6-sulfonic acid) (ABTS) free radical scavenging activity with half maximal effective concentration (EC50) of 23.2 ± 1.2 to 95.3 ± 0.6 μg/mL compared to ascorbic acid (IC50 = 38.72 μg/mL) [25].
Methanol extract of V. negundo leaves (100 – 1400 μg/mL) showed antioxidant activity with 2,2-azinobis(3-ethyl benzothiazoline-6-sulfonic acid) (ABTS) free radical scavenging activity with half maximal effective concentration (EC50) of 35.5 ± 0.9 to 95.8 ± 0.9 μg/mL compared to ascorbic acid (IC50 = 38.72 μg/mL) [25].
Ethanol extract of V. negundo leaves (100 – 1400 μg/mL) showed antioxidant activity with 2,2-azinobis(3-ethyl benzothiazoline-6-sulfonic acid) (ABTS) free radical scavenging activity with half maximal effective concentration (EC50) of 37.7 ± 1.3 to 96.7 ± 0.8 μg/mL compared to ascorbic acid (IC50 = 38.72 μg/mL) [ 25 ].
Ethyl acetate extract of V. negundo leaves (100 – 1400 μg/mL) showed antioxidant activity with 2,2-azinobis(3-ethyl benzothiazoline-6-sulfonic acid) (ABTS) free radical scavenging activity with half maximal effective concentration (EC50) of 11.2 ± 0.9 to 94.1 ± 1.2 μg/mL compared to ascorbic acid (IC50 = 38.72 μg/mL) [25].
HIV-1 reverse transcriptase inhibition activity
Ethanol extract of V. negundo leaves (200 μg/mL) inhibited the percentage of recombinant HIV-1 reverse transcriptase with inhibition rate of 92.8% compared to azidothymidine (inhibition rate: 85.37%) using HIV-1 reverse transcriptase assay [ 9 ].
Clinical studies
Information and data have not been established.
SAFETY INFORMATION
Preclinical studies (Toxicology studies)
Information and data have not been established.
Others (Adverse reaction, contraindication, side effect, warning, precaution)
Information and data have not been established.
DOSAGE
Information and data have not been established.
STORAGE
Store below 30°C. Protect from light and moisture.
REFERENCES
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