Anti-inflammatory activity of shikonin derivatives from Arnebia hispidissima.Summary Arnebia hispidissima Arnebia hispidissima poisonous plant in the family Boraginaceae containing the pyrrolizidine alkaloids monocrotaline and echimidine. ethanolic extract, after chromatography, yielded a number of shikonin derivatives, which were identified as arnebin-5, arnebin-6, teracryl shikonin, arnebinone and acetyl acetyl /ac·e·tyl/ (as´e-til) (as´e-tel?) (ah-se´til) the monovalent radical CH3COsbond, a combining form of acetic acid. a·ce·tyl n. shikonin. All these compounds were firstly reported from this plant species and evaluated to the anti-inflammatory activity of ethanolic extract and isolated shikonin derivatives, models with carrageenan-induced paw edema edema (ĭdē`mə), abnormal accumulation of fluid in the body tissues or in the body cavities causing swelling or distention of the affected parts. and complete Freund's adjuvant Freund's adjuvant n. A substance consisting of killed microorganisms, such as mycobacteria, in an oil and water emulsion that is administered to induce and enhance the formation of antibodies. (CFA)-induced chronic arthritis in rats were conducted. The observed results indicated that pre-treatment with arnebinone significantly inhibited the carrageenan-induced paw edema and also suppressed the development of chronic arthritis induced by CFA. Key words: Arnebia hispidissima, anti-inflammatory activity, shikonin derivatives * Introduction Arnebia hispidissima is annual herb and roots are sold in market under the trade name 'Ratanjot'. The herb possesses stimulant, tonic, diuretic diuretic (dī'yərĕt`ĭk), drug used to increase urine formation and output. Diuretics are prescribed for the treatment of edema (the accumulation of excess fluids in the tissues of the body), which is often the result of underlying and expectorant expectorant /ex·pec·to·rant/ (ek-spek´ter-ant) 1. promoting expectoration. 2. an agent that promotes expectoration. properties. The aqueous extract an extract obtained from a vegetable substance by steeping it in water. See also: Aqueous of flowering shoots are considered useful in diseases of tongue, throat and also in the treatment of fevers and cardiac disorders, (Anonymous, 1985; Satyavati et al. 1976), bruises, skin eruptions and arthritis (Jain and Defilipps, 1991). The roots yielded dl-alkannin (Shikalkin, yield-0.04%) as crystalline red oil (Jain and Mathur, 1965). Several arnebins, arnebin-1, arnebin-2, arnebin-3, arnebin-4, arnebin-5, arnebin-6 and arnebin-7 were isolated and characterized from A. nobilis (Shukla et al. 1969, 1971, 1973). Zhu et al. (1984) isolated shikonin and its five derivatives-deoxyshikonin, [beta], [beta]-dimethyl acetyl shikonin, acetyl shikonin, teracryl shikonin and [beta]-hydroxyisovaleryl shikonin from the roots of A. euchroma. Shikonin and its acetate derivatives as shikonin isovalerate isolated from A. decumbens (Mohammad and Galib, 1986a, b). The teracryl alkannin has found to be present in roots of A. densiflora. The triterpenic acids as 2 [alpha]-hydroxyursolic acid and tormentic acid have been isolated from A. euchroma (Yang et al. 1992). A. hispidissima yielded echimidine and monocrotaline (Gamila et al. 1987), [O.sup.9]-angeloyl retronecine and minor amount of [O.sup.7]-angeloyl retronecine isolated from A. euchroma (Roeder and Rengel-Mayer, 1993). Besides these reports, Arnebia species had antibacterial (Jain et al. 1999), anti-inflammatory (Lin et al. 1980; Tanaka et al. 1986), antitumor an·ti·tu·mor also an·ti·tu·mor·al adj. Counteracting or preventing the formation of malignant tumors; anticancer. Adj. 1. (Sankawa et al. 1977), anti-viral (Kashiwada et al. 1995) activities. But there are no reports in literature on A. hispidissima, for anti-inflammatory screening hence the study was carried out. The present study deals with the isolation and characterization of shikonin derivatives from A. hispidissima and were testing from intact plant species for anti-inflammatory activities. * Materials and Methods Plant material A. hispidissima (Lehm.) DC. (family-Boraginaceae) was collected (March, 1997) from fields near of Malviya Regional Engineering college, Jaipur and voucher specimens were deposited at the Herbarium herbarium, collection of dried and mounted plant specimens used in systematic botany. To preserve their form and color, plants collected in the field are spread flat in sheets of newsprint and dried, usually in a plant press, between blotters or absorbent paper. , Department of Botany, University of Rajasthan University of Rajasthan is the oldest university in the Indian state of Rajasthan.It was set up on 8 January 1947 as the University of Rajputana and was renamed to its current name in 1956. , Jaipur, India, were used for present investigation (sheet no. 19441) General experimental procedures Melting points were determined on capillary Toshniwal melting point melting point, temperature at which a substance changes its state from solid to liquid. Under standard atmospheric pressure different pure crystalline solids will each melt at a different specific temperature; thus melting point is a characteristic of a substance and apparatus and are uncorrected. The spectral data were obtained on the following instruments: ir, Perkin-Elmer, 283; uv, Perkin-Elmer, 200; ms, Hewlett Packard HP 5930 A; gc-ms, equipped with a HP 5933 data system, direct inlet at 70 ev; adsorbents for tlc (silica gel silica gel, chemical compound. It is a colloidal form of silica, and usually resembles coarse white sand. It may be prepared by partial dehydration of metasilicic acid, H2SiO3. Because it has many tiny pores, it has great adsorptive power. 60, 230-400 mesh; E Merck), tlc solvent system: hexane-benzene (3:1), hexane-acetone-acetic acid (15:5.0:0.3), MeOH-[H.sub.2]O (2:1), [C.sub.6][H.sub.6]: EtoAC-MeOH (3:1:1). The standards obtained from Y.N. Shukla, CIMAP CIMAP Central Institute for Medicinal and Aromatic Plants CIMAP Circuit Installation & Maintenance Package CIMAP Circuit Installation and Maintenance Assistance Package , Lucknow, India. Extraction and characterization Plant roots (10 kg) percolated with ethanol (95%) for 24 h, filtered and the resultant residue was Soxhlet extracted with ethanol (95%) for 24 h, filtered and concentrated. Both extracts were pooled together and subjected to further study (yield-l.98%). The ethanolic crude extract (180 g) was subjected to partition among hexane hexane /hex·ane/ (hek´san) a saturated hydrogen obtained by distillation from petroleum. hex·ane n. (Fr. I, 68.91 g), hexane-MeOH (2:1; Fr. II, 59.62 g) and CH[Cl.sub.3]-[H.sub.2]O (2:1; Fr. III, 35.38 g) which were used in subsequent work. The column chromatography column chromatography n. A form of partition chromatography in which a liquid phase flows down a column packed with a solid phase. of Fr. I on tlc grade silica gel (E Merck) with elution elution /elu·tion/ (e-loo´shun) in chemistry, separation of material by washing; the process of pulverizing substances and mixing them with water in order to separate the heavier constituents, which settle out in solution, from the by hexaneacetone; 6 fractions, A-F; Fr. II, elution by CH[Cl.sub.3]-[H.sub.2]O, 2 fractions, G-H; Fr. III with elution by EtoAC-MeOH yielded-3 fractions, I-K, were collected. Arnebin-5 (I) and arnebin-6 (II)--A portion of fraction A and B (23.28 g), were combined and rechromatographed on silica gel and purified by preparative pre·par·a·tive adj. Serving or tending to prepare or make ready; preliminary. n. Something that prepares for or acts as a preliminary to something following. tlc, with development by hexane-acetone-acetic acid, (15:5.0:0.3, [R.sub.f] ~ 0.13), followed by crystallization Crystallization The formation of a solid from a solution, melt, vapor, or a different solid phase. Crystallization from solution is an important industrial operation because of the large number of materials marketed as crystalline particles. with MeOH, yielded I (213 mg), mp 111[degrees]-112[degrees]C, [C.sub.16][H.sub.18][O.sub.5], red crystalline crystals (Shukla et al. 1971). The fraction C (15.62 g) was further separated and purified by means of preparative tlc II (168 mg), mp 88[degrees]-90[degrees]C, red crystalline crystals, with solvent system: hexane-acetone-acetic acid (15:5.0:0.3, [R.sub.f] ~ 0.16). These isolated compounds were subjected to various physical and spectral studies and identical with standard samples previously described (Shukla et al. 1969, 1971, 1973). Teracryl shikonin (III)--The fraction D-F, were combined together (22.34 g), purified by preparative tlc, solvent system: hexane-benzene, 3:1, [R.sub.f] ~ 0.21 and crystallized with methanol, red powder, yield-156 mg, [C.sub.23][H.sub.27][O.sub.7]. A direct comparison (ir, nmr and ms) of isolated compounds with an authentic sample of alkannin showed the two to be indistinguishable (Khan et al. 1983). Arnebinone (IV)--A portion of fraction G-H, pooled (48.31g), purified by preparative tlc (solvent mixture: methanol-water, 2:1, [R.sub.f] ~ 0.26) crystallized with MeOH, yield-248 mg, [C.sub.18][H.sub.22][O.sub.4], colourless colourless or US colorless Adjective 1. without colour: a colourless gas 2. dull and uninteresting: a colourless personality 3. needles, identified as arnebinone on the basis of ir. nmr, ms and uv of isolated arnebinone with authentic sample of arnebinone (Yao et al. 1991). Acetyl shikonin (V)--Isolated from fraction I-K (29.46 g), purified by preparative tlc (solvent system: [C.sub.6][H.sub.6]-EtoAC-MeOH, 3:1:1; v/v) crystallized in MeOH, yield-123 mg, [C.sub.18][H.sub.19][O.sub.7], red needles. Acetyl shikonin was identified by comparison (ir, nmr, ms and uv) with an authentic sample of acetyl shikonin showed the two to be indistinguishable (Zhu et al. 1984). Animals Carrageenan-induced inflammation in male Wistar rats weighing 150-200 g each and CFA-induced arthritis in male swiss albino albino (ălbī`nō) [Port.,=white], animal or plant lacking normal pigmentation. The absence of pigment is observed in the body covering (skin, hair, and feathers) and in the iris of the eye. mice (4-6 weeks old); weighing 25-30 g each) were used as model systems. The animals were obtained from Animal Centre, Hamdard University  The of this article or section may be compromised by "peacock terms".You can help Wikipedia by removing peacock terms. Hamdard University is located in Karachi, Sindh, Pakistan. , New Delhi New Delhi (dĕl`ē), city (1991 pop. 294,149), capital of India and of Delhi state, N central India, on the right bank of the Yamuna River. . All animals were housed in air conditioned room at 23[degrees] [+ or -] 2[degrees]C and fed with standard laboratory diet and tap water throughout the experiments. Carrageenan-induced paw edema in rats Anti-inflammatory test were performed according to according to prep. 1. As stated or indicated by; on the authority of: according to historians. 2. In keeping with: according to instructions. 3. the protocol of Winter et al. (1962). The carrageenan car·ra·geen·an or car·ra·geen·in n. Any of a group of closely related colloids derived from several red algae, widely used as a thickening, stabilizing, emulsifying, or suspending agent in pharmaceuticals. (Sigma St. Louis, MO) mixed with saline solution saline solution n. A solution of any salt, usually an isotonic sodium chloride solution. Also called salt solution. Saline solution A solution of sterile water and salt used in a variety of medical procedures. (2%, v/v) and 0.5 ml was injected subcutaneously into the right hind paw. The paw volume of each rat was measured before carrageenan injection and then hourly intervals upto 12 times by Plethysmometer 7150 (UGO UGO Underground Online (gaming website) , Basil, Italy). The drug groups (6 animals in each group) were treated was ethanolic extract, arnebin-5, arnebin-6, teracryl shikonin, amebinone and acetyl shikonin as isolated compounds (5, 10, 20 mg/kg body wt., s.c.) administered subcutaneously l h before carrageenan injection. The animals in the control group received saline only while other group of rats was administered subcutaneously with acetyl salicylic acid salicylic acid or 2-hydroxybenzoic acid, C6H4(OH)CO2H, a colorless, crystalline organic carboxylic acid that melts at 159°C;; it is soluble in ethanol and ether but is only slightly soluble in water. (5, 10, 20 mg/kg body wt., s.c.) in 1% ethanol, as standard reference. Adjuvant-induced arthritis in rats The adjuvant adjuvant /ad·ju·vant/ (aj?dbobr-vant) (a-joo´vant) 1. assisting or aiding. 2. a substance that aids another, such as an auxiliary remedy. 3. arthritis was induced by established method (Newbould, 1963). The left footpad footpad the thick, spongy structure located on each digit, and under the metacarpal- and metatarsal-phalangeal joints, and the carpus of dogs and cats. The skin is thickened, tough, and may be hyperpigmented and the hypodermis contains large amounts of adipose tissue. of each rat was injected subcutaneously with 0.05 ml of complete Freund's adjuvant (CFA, 1% suspension in olive oil olive oil, pale yellow to greenish oil obtained from the pulp of olives by separating the liquids from solids. Olive oil was used in the ancient world for lighting, in the preparation of food, and as an anointing oil for both ritual and cosmetic purposes. ; Difco). Rats in drug groups treated with ethanolic extract, arnebin-5, arnebin-6, teracryl shikonin, amebinone and acetyl shikonin as isolated compounds (5, 10, 20 mg/kg body wt., p.o.) 24 h before CFA injection and their daily treatment until 10 day after CFA challenge. The animals in control group received saline only. The another group of rats was administered with acetyl salicylic acid (5, 10, 20 mg/kg body wt., p.o.) in ethanol as standard reference. Statistical analysis Data were expressed as mean [+ or -] s.e.m as assessed statistically by analysis of variance (ANOVA anova see analysis of variance. ANOVA Analysis of variance, see there ). The difference between drug treated groups and control group was evaluated by Dunnett's 't' test. P < 0.01 was considered significant statistically. * Results and Discussion The A. hispidissima roots were extracted with EtOH and crude ethanolic extract after partitioning yield 68.91 g (hexane eluate eluate /el·u·ate/ (el´u-at) the substance separated out by, or the product of, elution or elutriation. el·u·ate n. The solution of solvent and dissolved matter resulting from elution. ), 59.62 g (hexane-MeOH; 2:1 eluate), 35.38 g (CH[Cl.sub.3]-[H.sub.2]O; 2:1 eluate) containing number of shikonin derivatives. These shikonin derivatives were separated from the fractions (I-K) by column chromatography on silica gel followed by preparative tlc. On the basis of spectral data and by direct comparison with authentic samples, these compounds were identified as arnebin-5 (I; 213 mg), amebin-6 (II; 168 g), teracryl shikonin (III; 156 mg), arnebinone (IV; 248 mg) and acetyl shikonin (V; 233 mg) (see Fig. 1). [FIGURE 1 OMITTED] Crude ethanolic extract, arnebin-5, arnebin-6, teracryl shikonin, amebinone and acetyl shikonin were investigated for anti-inflammatory activity. The pad of rat became edemateous soon after carrageenan injection and edema rate of the right hind paw reached it's peak at 8 h (1.95). The administration of crude extract and shikonin derivatives at the dose of 5, 10, 20 mg/kg body wt. significantly inhibited the development of paw swelling from 1 to 12 h after carrageenan injection (P < 0.01, Table 1). The observed results indicated that arnebinone showed moderate (27.17%) anti-inflammatory effects at the dose 5 mg/kg at 8h after carrageenan injection. The reference drug acetyl salicylic acid (5, 10, 20 mg/kg body wt.) was more effective than the extract and isolated compounds (37.94%, 8 h). The time course study of edema and inhibition rate after the administration of CFA and crude extract and isolated derivatives are depicted in Table 2. The edema rate was higher at 8 day and reached at it's peak (79.54%) and therefore the swelling subsided until the 10 day. The administration of crude ethanolic extract and shikonin derivatives at the dose of 5, 10, 20 mg/kg body wt. significantly inhibited the development of joint swelling induced by CFA. The anti-inflammatory effects of acetyl shikonin (64.42%) was higher as early as 2 day after CFA injection and was maintained until the experiment was terminated. The observed results provide a scientific basis for the utilization of this species in the treatment of variety of inflammatory activities. * Conclusions The observed results of this study, revealed that the ethanolic extract of the roots of A. hispidissima does have pronounced anti-inflammatory activity when used subcutaneously (Calvo et al. 1998). However, extracts only showed subcutaneous anti-inflammatory activity at a lower dosage far beyond that used as decoction DECOCTION, med. jurisp. The operation of boiling certain ingredients in a fluid, for the purpose of extracting the parts soluble at that temperature. Decoction also means the product of this operation. 2. or in a powdered form. The tested shikonin derivatives of the extract can be regarded totally or at least in part responsible for anti-inflammatory activity of the extract. The conclusions support scientific basis for the utilization of the plant in Indian folk medicine folk medicine, methods of curing by means of healing objects, herbs, or animal parts; ceremony; conjuring, magic, or witchcraft; and other means apart from the formalized practice of medical science. for the treatment of variety of subcutaneous inflammatory conditions.
Table 1. Anti-inflammatory effects of shikonin derivatives from
Arnebia hispidissima (Lehm.) DC. on carrageenan-induced paw edema.
Increased in foot edema
(ml) after injection
Treatment Dose (mg/kg) * 1 h
Control -- 1.59 [+ or -] 0.02
Ethanolic extract 50 1.42 [+ or -] 0.03 (a) (10.69)
100 1.39 [+ or -] 0.04 (a) (12.57)
150 1.31 [+ or -] 0.05 (a) (17.61)
Arnebin-5 5 1.28 [+ or -] 0.06 (a) (19.49)
10 1.26 [+ or -] 0.08 (b) (20.75)
20 1.30 [+ or -] 0.06 (a) (18.23)
Arnebin-6 5 1.27 [+ or -] 0.05 (a) (20.12)
10 1.33 [+ or -] 0.04 (b) (16.35)
20 1.34 [+ or -] 0.03 (a) (15.72)
Teracryl shikonin 5 1.44 [+ or -] 0.05 (a) (9.43)
10 1.36 [+ or -] 0.01 (a) (14.46)
20 1.41 [+ or -] 0.05 (a) (11.32)
Arnebinone 5 1.19 [+ or -] 0.04 (a) (25.15)
10 1.16 [+ or -] 0.03 (a) (27.04)
20 1.21 [+ or -] 0.02 (a) (23.89)
Acetyl shikonin 5 1.28 [+ or -] 0.01 (a) (19.49)
10 1.32 [+ or -] 0.06 (a) (16.98)
20 1.40 [+ or -] 0.05 (a) (11.94)
Acetyl salicylic acid 5 1.02 [+ or -] 0.03 (a) (35.84)
10 1.09 [+ or -] 0.02 (a) (31.44)
20 1.11 [+ or -] 0.02 (a) (30.18)
Increased in foot edema
(ml) after injection
Treatment Dose (mg/kg) * 2 h
Control -- 1.68 [+ or -] 0.05
Ethanolic extract 50 1.48 [+ or -] 0.06 (a) (11.90)
100 1.51 [+ or -] 0.05 (a) (10.11)
150 1.46 [+ or -] 0.04 (a) (13.09)
Arnebin-5 5 1.38 [+ or -] 0.01 (a) (17.85)
10 1.34 [+ or -] 0.02 (a) (20.23)
20 1.36 [+ or -] 0.03 (a) (19.04)
Arnebin-6 5 1.42 [+ or -] 0.05 (a) (15.47)
10 1.38 [+ or -] 0.05 (a) (17.85)
20 1.39 [+ or -] 0.04 (a) (17.26)
Teracryl shikonin 5 1.46 [+ or -] 0.03 (b) (13.09)
10 1.48 [+ or -] 0.01 (a) (11.90)
20 1.52 [+ or -] 0.02 (a) (9.52)
Arnebinone 5 1.26 [+ or -] 0.05 (a) (25.00)
10 1.23 [+ or -] 0.04 (a) (26.78)
20 1.29 [+ or -] 0.03 (a) (23.21)
Acetyl shikonin 5 1.34 [+ or -] 0.04 (a) (20.23)
10 1.38 [+ or -] 0.05 (a) (17.85)
20 1.43 [+ or -] 0.04 (a) (14.88)
Acetyl salicylic acid 5 1.08 [+ or -] 0.08 (a) (35.71)
10 1.12 [+ or -] 0.09 (b) (33.33)
20 1.15 [+ or -] 0.01 (a) (18.77)
Increased in foot edema
(ml) after injection
Treatment Dose (mg/kg) * 4 h
Control -- 1.79 [+ or -] 0.02
Ethanolic extract 50 1.58 [+ or -] 0.03 (a) (11.73)
100 1.63 [+ or -] 0.04 (a) (8.93)
150 1.52 [+ or -] 0.01 (a) (15.08)
Arnebin-5 5 1.42 [+ or -] 0.02 (a) (20.67)
10 1.46 [+ or -] 0.01 (a) (18.43)
20 1.39 [+ or -] 0.01 (a) (22.34)
Arnebin-6 5 1.46 [+ or -] 0.03 (a) (18.43)
10 1.54 [+ or -] 0.02 (a) (13.96)
20 1.52 [+ or -] 0.01 (a) (15.08)
Teracryl shikonin 5 1.60 [+ or -] 0.03 (a) (10.61)
10 1.67 [+ or -] 0.05 (a) (6.70)
20 1.61 [+ or -] 0.07 (a) (10.5)
Arnebinone 5 1.35 [+ or -] 0.08 (a) (24.58)
10 1.41 [+ or -] 0.09 (a) (21.22)
20 1.48 [+ or -] 0.08 (a) (17.31)
Acetyl shikonin 5 1.42 [+ or -] 0.05 (a) (20.67)
10 1.45 [+ or -] 0.04 (a) (18.99)
20 1.49 [+ or -] 0.03 (a) (16.75)
Acetyl salicylic acid 5 1.19 [+ or -] 0.02 (b) (33.51)
10 1.22 [+ or -] 0.01 (a) (31.84)
20 1.28 [+ or -] 0.04 (a) (28.49)
Increased in foot edema
(ml) after injection
Treatment Dose (mg/kg) * 8 h
Control -- 1.95 [+ or -] 0.05 (a)
Ethanolic extract 50 1.7 [+ or -] 0.04 (a) (12.30)
100 1.75 [+ or -] 0.03 (a) (10.25)
150 1.68 [+ or -] 0.03 (a) (13.84)
Arnebin-5 5 1.51 [+ or -] 0.02 (a) (22.56)
10 1.55 [+ or -] 0.05 (b) (20.51)
20 1.41 [+ or -] 0.04 (a) (27.69)
Arnebin-6 5 1.54 [+ or -] 0.04 (a) (21.02)
10 1.59 [+ or -] 0.05 (a) (18.46)
20 1.60 [+ or -] 0.06 (a) (17.94)
Teracryl shikonin 5 1.49 [+ or -] 0.08 (a) (23.58)
10 1.69 [+ or -] 0.07 (a) (13.33)
20 1.76 [+ or -] 0.06 (a) (9.74)
Arnebinone 5 1.42 [+ or -] 0.01 (a) (27.17)
10 1.46 [+ or -] 0.02 (a) (25.12)
20 1.59 [+ or -] 0.01 (a) (18.46)
Acetyl shikonin 5 1.55 [+ or -] 0.05 (a) (20.51)
10 1.56 [+ or -] 0.06 (a) (20.00)
20 1.51 [+ or -] 0.02 (a) (22.56)
Acetyl salicylic acid 5 1.21 [+ or -] 0.01 (a) (37.94)
10 1.26 [+ or -] 0.02 (b) (35.38)
20 1.34 [+ or -] 0.04 (a) (31.28)
Increased in foot edema
(ml) after injection
Treatment Dose (mg/kg) * 12 h
Control -- 1.89 [+ or -] 0.01 (a)
Ethanolic extract 50 1.62 [+ or -] 0.02 (a) (14.28)
100 1.72 [+ or -] 0.01 (a) (8.99)
150 1.65 [+ or -] 0.02 (a) (12.69)
Arnebin-5 5 1.61 [+ or -] 0.02 (b) (14.81)
10 1.53 [+ or -] 0.04 (a) (19.04)
20 1.40 [+ or -] 0.02 (a) (25.92)
Arnebin-6 5 1.56 [+ or -] 0.01 (a) (17.46)
10 1.63 [+ or -] 0.04 (a) (13.75)
20 1.68 [+ or -] 0.05 (a) (11.11)
Teracryl shikonin 5 1.58 [+ or -] 0.05 (a) (16.40)
10 1.60 [+ or -] 0.06 (a) (15.34)
20 1.75 [+ or -] 0.07 (a) (7.40)
Arnebinone 5 1.52 [+ or -] 0.09 (a) (19.57)
10 1.59 [+ or -] 0.08 (a) (15.87)
20 1.67 [+ or -] 0.04 (a) (11.64)
Acetyl shikonin 5 1.56 [+ or -] 0.06 (a) (17.46)
10 1.62 [+ or -] 0.05 (a) (10.58)
20 1.69 [+ or -] 0.04 (a) (10.58)
Acetyl salicylic acid 5 1.19 [+ or -] 0.03 (a) (37.03)
10 1.34 [+ or -] 0.01 (b) (29.10)
20 1.39 [+ or -] 0.02 (a) (26.45)
* mg/kg body weight; values represent the mean [+ or -] S.E.M. of six
animals for each groups; statistically significant from control
(a) P < 0.01 and (b) P < 0.05 (Dunnett's 't' test).
Each value in parenthesis indicates the percentage inhibition rate.
Table 2. Anti-inflammatory effects of shikonin derivatives from A
hispidissima (Lehm.) DC. on CFA-induced paw edema in rats.
Edema rate (%)
Group Dose 1 day
(mg/kg) *
Control -- 36.52 [+ or -] 3.31
Ethanolic 50 17.18 [+ or -] 1.16 (a) (52.95)
extract 100 21.56 [+ or -] 1.23 (b) (40.96)
150 24.50 [+ or -] 2.34 (a) (32.91)
Arnebin-5 5 22.69 [+ or -] 3.38 (a) (37.86)
10 19.45 [+ or -] 4.52 (a) (46.74)
20 20.61 [+ or -] 6.61 (a) (43.56)
Arnebin-6 5 25.43 [+ or -] 3.39 (b) (30.36)
10 28.65 [+ or -] 2.12 (a) (21.54)
20 31.39 [+ or -] 1.08 (b) (14.04)
Teracryl 5 24.26 [+ or -] 1.19 (b) (33.57)
shikonin 10 22.16 [+ or -] 2.16 (a) (39.32)
20 19.56 [+ or -] 3.34 (a) (46.44)
Arnebinone 5 13.71 [+ or -] 3.52 (a) (62.45)
10 16.54 [+ or -] 4.41 (a) (54.70)
20 14.12 [+ or -] 5.56 (a) (61.33)
Acetyl 5 18.62 [+ or -] 3.54 (a) (49.0)
shikonin 10 21.43 [+ or -] 4.41 (b) (41.31)
20 22.68 [+ or -] 3.39 (a) (37.89)
Acetyl 5 10.21 [+ or -] 2.82 (a) (72.04)
salicylic 10 11.19 [+ or -] 1.69 (a) (69.35)
acid 20 13.52 [+ or -] 1.19 (b) (62.97)
Edema rate (%)
Group Dose 2 days
(mg/kg) *
Control -- 49.61 [+ or -] 4.42
Ethanolic 50 20.22 [+ or -] 2.16 (a) (59.24)
extract 100 26.29 [+ or -] 3.31 (a) (47.00)
150 24.61 [+ or -] 1.19 (b) (50.39)
Arnebin-5 5 28.31 [+ or -] 2.69 (a) (42.93)
10 26.49 [+ or -] 1.16 (a) (46.60)
20 24.31 [+ or -] 1.28 (a) (50.99)
Arnebin-6 5 26.71 [+ or -] 1.31 (a) (46.16)
10 31.32 [+ or -] 4.47 (a) (36.86)
20 34.59 [+ or -] 3.31 (a) (30.27)
Teracryl 5 37.52 [+ or -] 2.19 (a) (24.37)
shikonin 10 30.60 [+ or -] 8.60 (b) (38.31)
20 34.30 [+ or -] 7.52 (a) (30.86)
Arnebinone 5 36.54 [+ or -] 4.32 (a) (26.34)
10 20.41 [+ or -] 4.30 (b) (58.85)
20 19.52 [+ or -] 2.39 (a) (60.65)
Acetyl 5 17.65 [+ or -] 2.20 (a) (64.42)
shikonin 10 28.21 [+ or -] 1.19 (a) (43.13)
20 32.31 [+ or -] 2.68 (b) (34.87)
Acetyl 5 34.54 [+ or -] 3.39 (a) (30.37)
salicylic 10 16.50 [+ or -] 4.42 (a) (66.74)
acid 20 14.31 [+ or -] 3.31 (a) (71.15)
Edema rate (%)
Group Dose 4 days
(mg/kg) *
Control -- 58.42 [+ or -] 2.68
Ethanolic 50 29.61 [+ or -] 2.61 (a) (49.31)
extract 100 31.32 [+ or -] 3.34 (a) (46.38)
150 33.42 [+ or -] 2.89 (a) (42.93)
Arnebin-5 5 39.46 [+ or -] 1.69 (a) (32.45)
10 41.52 [+ or -] 1.11 (a) (28.92)
20 39.46 [+ or -] 1.32 (a) (32.45)
Arnebin-6 5 34.52 [+ or -] 1.98 (b) (40.91)
10 38.66 [+ or -] 3.34 (a) (33.82)
20 42.61 [+ or -] 4.49 (a) (27.06)
Teracryl 5 44.66 [+ or -] 5.59 (a) (23.55)
shikonin 10 43.53 [+ or -] 6.40 (a) (25.48)
20 48.66 [+ or -] 3.31 (a) (16.70)
Arnebinone 5 28.61 [+ or -] 2.68 (b) (51.02)
10 26.54 [+ or -] 3.39 (a) (54.48)
20 24.51 [+ or -] 4.41 (a) (58.04)
Acetyl 5 31.50 [+ or -] 6.61 (a) (46.08)
shikonin 10 37.89 [+ or -] 6.68 (a) (35.14)
20 4.44 [+ or -] 17.72 (b) (29.06)
Acetyl 5 19.35 [+ or -] 4.46 (a) (66.87)
salicylic 10 16.53 [+ or -] 5.69 (a) (71.70)
acid 20 21.44 [+ or -] 6.89 (a) (63.30)
Edema rate (%)
Group Dose 6 days
(mg/kg) *
Control -- 69.51 [+ or -] 3.31
Ethanolic 50 34.45 [+ or -] 3.39 (a) (50.43)
extract 100 38.61 [+ or -] 3.68 (a) (44.45)
150 39.41 [+ or -] 4.49 (a) (43.30)
Arnebin-5 5 44.61 [+ or -] 5.19 (b) (35.82)
10 42.31 [+ or -] 5.68 (a) (39.13)
20 46.39 [+ or -] 4.43 (a) (33.26)
Arnebin-6 5 41.34 [+ or -] 4.41 (a) (40.52)
10 46.45 [+ or -] 3.78 (b) (33.17)
20 51.31 [+ or -] 2.68 (a) (26.18)
Teracryl 5 59.53 [+ or -] 3.3 (a) (14.35)
shikonin 10 49.31 [+ or -] 2.19 (a) (29.06)
20 58.21 [+ or -] 1.89 (a) (16.25)
Arnebinone 5 34.34 [+ or -] 1.19 (b) (50.59)
10 36.51 [+ or -] 2.68 (a) (47.47)
20 33.54 [+ or -] 3.13 (a) (51.74)
Acetyl 5 41.41 [+ or -] 4.46 (a) (40.42)
shikonin 10 44.61 [+ or -] 3.38 (b) (35.82)
20 51.54 [+ or -] 5.56 (a) (25.85)
Acetyl 5 26.35 [+ or -] 4.41 (a) (62.09)
salicylic 10 21.43 [+ or -] 3.39 (a) (69.16)
acid 20 26.53 [+ or -] 2.61 (a) (61.83)
Edema rate (%)
Group Dose 8 days
(mg/kg) *
Control -- 79.54 [+ or -] 1.19
Ethanolic 50 41.44 [+ or -] 4.39 (a) (47.90)
extract 100 44.65 [+ or -] 4.39 (a) (43.86)
150 46.56 [+ or -] 4.41 (a) (41.46)
Arnebin-5 5 48.31 [+ or -] 4.42 (a) (39.26)
10 51.21 [+ or -] 3.19 (a) (35.61)
20 54.22 [+ or -] 2.69 (a) (31.83)
Arnebin-6 5 48.44 [+ or -] 2.19 (a) (39.09)
10 51.56 [+ or -] 16.16 (a) (35.17)
20 58.21 [+ or -] 5.55 (a) (26.81)
Teracryl 5 61.23 [+ or -] 4.44 (b) (23.0)
shikonin 10 59.61 [+ or -] 3.59 (a) (25.05)
20 62.61 [+ or -] 2.61 (b) (21.28)
Arnebinone 5 36.21 [+ or -] 3.44 (a) (54.47)
10 39.61 [+ or -] 33.9 (a) (50.20)
20 34.54 [+ or -] 2.69 (a) (56.57)
Acetyl 5 46.85 [+ or -] 5.61 (b) (41.09)
shikonin 10 49.44 [+ or -] 4.34 (a) (37.84)
20 58.61 [+ or -] 5.61 (a) (26.31)
Acetyl 5 31.41 [+ or -] 4.44 (a) (60.51)
salicylic 10 38.21 [+ or -] 3.39 (a) (51.96)
acid 20 39.61 [+ or -] 3.30 (b) (50.20)
Edema rate (%)
Group Dose 10 days
(mg/kg) *
Control -- 76.52 [+ or -] 4.44
Ethanolic 50 42.3 [+ or -] 1.24 (a) (44.70)
extract 100 41.31 [+ or -] 1.16 (a) (46.01)
150 44.31 [+ or -] 2.68 (a) (42.09)
Arnebin-5 5 46.51 [+ or -] 1.19 (a) (39.21)
10 49.15 [+ or -] 1.15 (a) (35.76)
20 51.51 [+ or -] 2.18 (a) (32.68)
Arnebin-6 5 46.54 [+ or -] 3.23 (a) (39.17)
10 49.31 [+ or -] 3.30 (a) (35.55)
20 54.54 [+ or -] 1.89 (b) (28.72)
Teracryl 5 59.61 [+ or -] 1.19 (b) (22.10)
shikonin 10 58.61 [+ or -] 2.68 (a) (23.39)
20 64.16 [+ or -] 2.16 (a) (16.15)
Arnebinone 5 41.40 [+ or -] 1.16 (a) (45.89)
10 36.61 [+ or -] 2.42 (a) (52.15)
20 39.42 [+ or -] 4.40 (b) (48.48)
Acetyl 5 44.63 [+ or -] 4.41 (a) (41.67)
shikonin 10 51.42 [+ or -] 4.39 (a) (32.80)
20 56.41 [+ or -] 2.19 (a) (26.28)
Acetyl 5 36.41 [+ or -] 3.39 (b) (52.41)
salicylic 10 39.31 [+ or -] 2.19 (a) (48.62)
acid 20 41.31 [+ or -] 2.69 (a) (46.01)
* mg/kg body weight; values represent the Mean [+ or -] S.D. of six
animals in each groups; statistically significant from control
(a) P < 0.01 and (b) P < 0.05 (Dunnett's 't' test).
Each value in parenthesis indicates the percentage inhibition rate.
Acknowledgements Authors are thankful to Professor Naveen Sharma, National Institute of Ayurveda, Jaipur for his help in anti-inflammatory activity, Dr. M.H. Khan for statistical analysis, Department of Statistics, Agricultural Research Station, Durgapura, Jaipur and Department of Biotechnology The Centre for Biotechnology at Acharya Nagarjuna University was established in year 1994 inaugurated by the then Secretary, Department of Biotechnology, Government of India, Dr.C.R.Bhatia. The centre was offering two academic programs, M.Sc. (Biotechnology) and M.Tech. , New Delhi, for financial support. * Reference Anonymous (1985) The Wealth of India-Raw Materials. PID (1) (Process IDentifier) A temporary number assigned by the operating system to a process or service. (2) (Proportional-Integral-Derivative) The most common control methodology in process control. , CSIR CSIR Council for Scientific and Industrial Research (Ghana) CSIR Council of Scientific and Industrial Research (India) CSIR Centre for Scientific and Industrial Research , New Delhi, pp 429-430 Calvo MI, Vilalta N, San Julian A, Fernandez M (1998) Antiinflammatory activity of leaf extract of Verbena officinalis Verbena officinalis, n See European vervain. L. Phytomedicine 5: 465-467 Gamila W, Bassem EM, Atta S, Gama M (1987) Toxic pyrrolizidine alkaloids alkaloids, n alkaline phytochemicals that contain nitrogen in a heterocyclic ring structure. They can have powerful pharmacological effects and are more often used in traditional medicine than in herbal treatments. of certain Boraginaceous plants. Acta Pharm Seuc 24: 199-204 Jain AC, Mathur SK (1965) A chemical study of the pigments of Arnebia hispidissima. Bull Nat Inst Sci India 28: 52-56 Jain SC, Singh B, Jain R (1999) Arnebins and antimicrobial antimicrobial /an·ti·mi·cro·bi·al/ (-mi-kro´be-al) 1. killing microorganisms or suppressing their multiplication or growth. 2. an agent with such effects. activity of Arnebia hispidissima (Lehm.) DC. cell cultures. Phytomedicine 6: 474-476 Jain SK, Defilipps RA (1991) Medicinal Plants medicinal plants, plants used as natural medicines. This practice has existed since prehistoric times. There are three ways in which plants have been found useful in medicine. of India. Reference Publ Inc Michigan, pp 94 Kashiwada Y, Nishizawa M, Yamagishi T, Tanaka T, Nonaka G-I, Cosentino LM, Snider JV, Lee KH (1995) Anti-AIDS agents 18. Sodium and potassium salts of caffeic acid an acid obtained from coffee tannin, as a yellow crystalline substance, See also: Caffeic tetramers from Arnebia euchroma as anti-HIV agents. J Nat Prod 58: 392-400 Khan HA, Chandrashekharan I, Ghanim A (1983) Naphthazarines from Arnebia hispidissima. Phytochemistry phytochemistry, n the scientific study and classification of the chemical constituents of plants. 22: 614-615 Lin ZB, Chai BL, Wang P, Guo QX, Lu FS, Xiang GQ (1980) Studies on the anti-inflammatory effect of chemical principle of Zi-cao (Arnebia euchroma). Pei Ching I Hsueh Yuan Hsueh Pao 12: 101-106 Mohammad A, Galib A-O (1986a) Shikonin derivatives IV. Chemical investigation of Arnebia decumbens. Agri Biol Chem 50: 1651-1652 Mohammad A, Galib A-O (1986b) Shikonin derivatives V. Chemical investigation of Arnebia decumbens. Agri Biol Chem 50: 759-760 Newbould BB (1963) Chemotherapy of arthritis induced in rats by mycobacterial mycobacterial emanating from or pertaining to mycobacterium. mycobacterial granuloma may be caused by Mycobacterium tuberculosis (see cutaneous tuberculosis), M. adjuvant. British J Pharmacol 45: 113-123 Roeder E, Rengel-Mayer B (1993) Pyrrolizidine alkaloids from Arnebia euchroma. Planta Med 59: 192 Sankawa U, Ebizuka Y, Miyazaki T, Isomura Y, Otsuka H, Shibata S, Inomata M, Fukuoka F (1977) Antitumor activity of shikonin and its derivatives. Chem Pharm Bull 25: 2392-2395 Satyavati GV, Raina MK, Sharma M (1976) Medicinal Plants of India. ICMR ICMR Indian Council of Medical Research ICMR Institute for Coastal and Marine Resources ICMR Interagency Committee on Medical Records , New Delhi, pp 92 Shukla YN, Tandon JS, Bhakuni DS, Dhar MM (1969) Chemical constituents of the antibiotic fraction of Arnebia nobilis. Experientia 25: 357-358 Shukla YN, Tandon JS, Bhakuni DS, Dhar MM (1971) Naphthaquinones of Arnebia nobilis. Phytochemistry 10: 1909-1915 Shukla YN, Tandon JS, Bhakuni DS, Dhar MM (1973) Arnebin-7, a new naphthaquinone from Arnebia nobilis. Indian J Chem 11: 528-529 Tanaka S, Kuwai Y, Staba M (1986) A comparative study on anti-inflammatory activities of enantiomers enantiomers (i·nanˑ·tē·  ·merz),n. , shikonin and alkannin. J Nat Prod 49: 466-469 Winter CA, Risley EA, Nuss CW (1962) Carrageenan-induced edema in hind paw of the rats as an assay for anti-inflammatory drugs. Proc Soc Exp Biol Med 111: 544-547 Yang MH, Blunden GO, Neil MJ, Lewis JA (1992) Tormentic acid and 2 [alpha]-hydroxyursolic acid from Arnebia euchroma. Planta Med 58: 227 Yao XS, Ebizuka Y, Noguchi H, Kiuchi F, Shibuya M, Iitaka Y, Seto H, Sankawa U (1991) Biologically active constituents of Arnebia euchroma: Arnebinone and arnebifuranone. Chem Pharm Bull 39: 2962-2964 Zhu F, Lu F, Xiang G (1984) Isolation of shikonin and its derivatives by HPLC HPLC high-performance liquid chromatography. HPLC high performance liquid chromatography. HPLC High-performance liquid chromatography Lab instrumentation A highly sensitive analytic method in which analytes are placed . Sepu 1: 131-133 * Address Swaroop Singh, Department of Entomology entomology, study of insects, an arthropod class that comprises about 900,000 known species, representing about three fourths of all the classified animal species. , Agriculture Research Station, Durgapura, Jaipur--302018, India Tel.: ++91-141-2722054; Fax: ++91-141-624642; e-mail: priyankasharma@id.eth.net B. Singh (1), M. K. Sharma (1), P. R. Meghwal, P. M. Sahu (1), and S. Singh (2) (1) Department of Botany, University of Rajasthan, Jaipur, India (2) Department of Entomology, Agriculture Research Station, Durgapura, Jaipur, India |
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