stoechas L.

Scientific Name: Lavandula stoechas L. Synonyms: Stoechas officinarum Mill. Family: Genus: Lavandula : stoechas Common name: French Lavender, Spanish Lavender, Stoechas Lavender, or Topped Lavender Part used: , Dried and Flower oil Description: An evergreen shrub, it usually grows to 30–100 cm (12–39 in) tall and occasionally up to 2 m (7 ft) high in the subspecies luisieri. The leaves are 1–4 cm long, greyish and tomentose. The are pink to purple, produced on spikes 2 cm long at the top of slender, leafless stems 10–30 cm (4–12 in) long; each flower is subtended by a 4–8 mm long. At the top of the spike are a number of much larger, sterile (no flowers between them), 10–50 mm long and bright lavender purple. Chemical constituents: Lavender flower contains 1.5-3% volatile oil of which 25-55% is linalyl acetate, 20-38% linalool, 4-10% cis linalyl acetate, 2-6% trans β- ocimene, 2-6% 1-terpinen-4-ol, 2% 3-octanone, 0.3-1.5% 1-8-cineole, 0.3-1% α-terpineol, 0.2-0.5% camphor and 0.1- 0.5%limonene, tannins 5-10%, hydroxyl coumarins, umbelliferone, herniarin, flavonoids (luteolin), phytosterols and triterpenes. Caffeic acid derivatives include rosmaric acid. Structures of chemical constituents of Lavandula stoechas L.

Linalyl acetate Linalool Trans Ocimene Terpinen-4-ol

3-octanone 1-8-cineole α-terpineol camphor

303

Limonene Umbelliferone Herniarin

Luteolin

Action of Herb: Anti-microbial, anti-inflammatory, anti-convulsant, anti-spasmodic, insecticidal, hypoglycaemic activity, anti-oxidant, expectorant, carminative, stimulant, deobstruent, resolvent, wound healing, analgesic, muscle relexant, sedative, anxiolytic effect. Medicinal uses: It is used for pain of head and brain that are due to cold, falling sickness, dropsy, cramps, convulsions, palsies and fainting. It is stomach tonic and used in liver and spleen obstructions. It is nervine tonic. Germans use it for loss of appetite, nervousness and insomnia. It is used in depression, anxiety, irritability, epilepsy, palsy, hemiplegia and cephalgia. The alcoholic extract produced 61.66%, crude powder produced 33.3%, and aqueous extract produced 16.66% response against sinusitis. Inhalation of lavender oil vapor causes dose dependent suppression of convulsions in mice by PTZ, nicotine and electric shock. Aromatherapy with lavender oil increase sleep time comparable to benzodiazepines and neuroleptics. It is also used in catarrh stomach irritation, headache with constipation. Side Effects: Frequent use of lavender cause colic and griping. Contraindications: Pregnancy Dosage:1-2 tea spoons (3-5g) of dried herb per cup of tea or 1-4 drops of lavender oil with sugar or 750mg tablet, 2 tablets 3 times for 3 weeks treat sinusitis is recommended. Chemical Identification Tests: The presence of different chemical constituents in flower of L. stoechas was identified by reactions with various chemical reagents. The reactions showed positive results for the presence of triterpenes, tannin, alkaloids, carbohydrate and sterols, while the test for protein is negative.

304 Thin-layer Chromatography of L. stoechas extract: The result of TLC of L. stoechas in ethylacetate-methanol-water solvent system in the ratio of 100:16.5:13.5 (254: 0.21, 0.43, 0.58, 0.61; 366: 0.18, 0.36, 0.66 & 0.8) and chloroform- methanol-water in the ratio of 80:20:2 (254: 0.53, 0.62, 0.81, 0.87; 366: 0.32, 0.41, 0.48) solvent systems. The TLC plates were observed under UV light at 254 and 366 nm. The distances of spots are measured after spraying with Vanillin-Sulphuric acid reagent. Phytochemical screening of L. stoechas extract All the extracts of L. stoechas revealed the presence of significant amount of phenols, flavonoids and terpenes and absence of alkaloids. Table: Phytochemical examination of L. stoechas extract

Histology and Histo-chemistry of L. stoechas extract In L. stoechas subsp. luisieri their stalk was up to 52-85 mm long. In the lower epidermis this type of was denser and shorter, up to 26-39 mm in L. stoechas subsp. luisieri . in L. stoechas subsp. luisieri , capitate type I with 1 basal cell, 1stalk cell and a head with 1-2 broad cells. capitate type II with 1 conical basal cell, 1-2 stalk cells and a round apical head.

Figure 1: Transverse section of showing upper epidermis with non-glandular, multi-cellular branched trichomes with a stalk bearing stellate type several arms

305

Figure 2: Capitate type IFigure 3: Capitate type II Histo-chemical analysis of L. stoechas extract revealed following observations:

Figure 4: [A] Peltate trichomes stained with Nile blue; [B] Peltate stained with 2,4 Dinitro- phenilhidraze; [C] Capitate trichomes stained positive with Sudan red; [D] Capitate type I trichome stained positive with Rubeanic acid. Fourier Transform Infra-red Spectroscopy of L.Stoechas extract: Lavendula Stoechas extract revealed prominent peaks of OH, CH, aromatic ring, C=C, C-O-C.

306

High Performance Liquid Chromatography of Lavandula Stoechas extract

Analgesic activity: In acetic acid induced writhing test aspirin 300 mg/kg was used as a reference compound. The crude extract of L. stoechas showed significant dose related inhibition of number of writhes. Table 2a showed that in vehicle treated (control) animals, mean number of writhes induced by intraperitoneal injection of acetic acid were 135, which was reduced to 40, 59 and 76 in animals treated with 500, 300 and 200 mg/kg oral dose of the test substance respectively. Table 2a revealed that the results of the writhing test were highly significant and comparable with aspirin which produced 48 writhes. The % of inhibition of writhes with the three doses of crude extract were 70.4% (500mg/kg) 56.3% (300mg/kg) and 43.7% (200mg/kg) whereas with aspirin it was 64.4% (300 mg/kg). The results of hot plate activity were presented in table 2b. The readings were observed for 3 hours. It showed slight dose dependent increase in tail flick time. At 500 mg/kg it showed 0.38 second flicking time at 2 hour whereas aspirin 300 mg/kg produced significant delayed in pain response at 2 hour (0.43). Table: Acetic acid induced writhing test Treatment Dose(mg/kg) Mean No. of writhes % of inhibition Control 0.5ml saline 128±3.48 - Crude Extract 500 63±3.36 50.1* Crude Extract 300 55 ±2.16 57.03 Crude Extract 200 43±1.96 66.41 Aspirin 300 46.4±1.27 63.8* Each value is the mean ± S.E.M. of five determinations. * P < 0.05, Dunnet test as compared to control

Table: Hot plate activity of crude extract of Lavendula stoechas, variation flicking time (s) Group 0 1 2 3 Control 0.19±0.014 >0.22 >0.22 >0.22 500 0.18±0.04 0.38±0.05 0.45±0.06 0.43±0.01* 300 0.20±0.03 0.34±0.02 0.39±0.04 0.38±0.04 200 0.20±0.06 0.31±0.04 0.38±0.04 0.34±0.07 100 0.19±0.04 0.24±0.03 0.21±0.02 0.22±0.05 Aspirin 0.18±0.02 0.39±0.03* 0.44±0.04* 0.33±0.08 Each value is the mean ± S.E.M. of five determinations. * P < 0.05, Dunnet test as compared to control

307 Neuro-pharmacological Studies on L. stoechas The gross behaviour activity was observed for both the control and test substance. Crude extract of L. stoechas at the three different doses i.e. 200mg kg, 300mg/kg and 500mg/kg for at least 24 hours. The results are mentioned in table 3a.The same procedure was followed for the standard drugs Diazepam 2 mg/kg and Imipramine 15 mg/kg. Table 3b exhibits the mean number of squares crossed by the mice were 121 for control group, 60 for 500mg/kg, 74 for 300mg/kg and 88 for 200mg/kg which showed that L. stoechas causes CNS Depressant action. The Diazepam (2mg/kg) and Imipramine (15mg/kg) treated groups showed 31 and 166 squares traveled respectively. The results of head dip, cage cross and rearing activities were presented in table 3b. There was decreased in the head dip and cage cross and rearing activity. In head dip test they showed 11, 21 and 30 times response for 500, 300 and 200 mg/kg respectively (control group: 47, Diazepam treated group: 05, Imipramine treated: 45). The results of cage cross activity were 53, 16, 22, 34, 09, 39 respectively for control, 500, 300, 200, 2(Diazepam) and 15 (Imipramine) mg/kg treated groups. Similarly results of rearing activity also showed decreased in movements. The readings of rearing activity were 42, 20, 34, 39, 14, 55 respectively for control, 500, 300, 200, 2 (Diazepam) and 15 (Imipramine) mg/kg treated groups. Table 3b showed that the time taken to travel iron rod was significantly increased at the dose of 500mg/kg, 300 and 200 mg/kg which was 300, 160 and 60 seconds respectively while it was 180 seconds for Diazepam and 10 seconds for Imipramine. Table 3b showed that the mean mobility time in water tub of animals with L. stoechas was 4.1, 2.11, 2.44, 3.1, 2.1 and 4.4 minutes respectively for control, 500, 300, 200, 2 (Diazepam) and 15 (Imipramine) mg/kg treated groups.

Table: Behavioral profile of Lavendula stoechas Parameters Doses (mg/kg) Diazepam Imipramine 500 300 200 100 50 25 10 1 2mg/kg 10mg/kg Nystagmus +2 +2 +2 +1 + - - - +1 - Vocalization ------Piloerection +1 +1 ------+ Micturition +2 +1 + + + + +s +s +s + Irritability + + - - - + + + +s + Disorientation/Staggeringgate + ------+ Aggressiveness +1 + +s ------+s Motor activity (Decrease) +2 +2 + + + + + + + - Pain response + + + + + + + + + + Ataxia ------+ Tail erection + + ------+ Tremor +2 +1 ------+2

308 Table: Assesment of neuro-pharmacological activities Lavendula stochoes Treatment Dose mg/kg Open field Head dip Cage cross Rearing test Mobility Traction orally time (min) test (sec) Control 0.5ml 121.8±4.6 34±3.75 35.7±1.54 39.17±0.93 3.45±1.12 22 ±1.05 Crude Extract 500 43±4.15 18±1.56 21±0.56 32.17±1.51 2.21±1.45 50±1.07* Crude Extract 300 56±2.34 26±1.84 27± 1.58 28± 0.51 2.16±1.03 40±1.02 Crude Extract 200 69±3.34 28± 2.14 29±2.71 22±1.52 2.74±1.01 33±1.13 Diazepam 2 31±1.12 05±0.09 09±1.12 14±1.12 2.1±1.3 180±3.12 Imipramine 15 166±4.2 45±1.42 39±2.3 55±2.5 4.4±2.1 06±0.09 Mean number of observation ± SEM* at P < 0.05 and P > 0.05* significant, **moderate significant, ***highly significant GC-MS analysis of volatile oil present in L. stoechas L.stoechas contains volatile oil enriched with 42 constituents. High occurrence of pulegone, menthol, menthone, fenchone and pinocarvyl acetate is usually observed.

Table: GC-MS analysis of total volatile content present in L. stoechas

Gören AC, Top çu G, Bilsel G, Bilsel M, Aydo ğmu ș Z, Pezzuto JM (2002). The chemical constituents and biological activity of essential oil of Lavandula stoechas ssp. Stoechas. Z. Naturforsch., 57c: 797-800.

309 Effects of Lavandula stoechas on healing of experimental skin wounds L. stoechas extract was compared with zinc oxide ointment in the experiment of open surgical skin wound healing in the rats. Macroscopic and microscopic study of healing process was studied at 0, 3, 7, 14, 21 and 28 th days of experiment in all of the rats. Maximum healing potential was observed in 10% L. stoechas extract (p <0.05) better than other experimental groups and the control group (zinc oxide topical ointment).

References Angioni A, Barra A, Coroneo V, Dessi S, Cabras P. 2006. Chemical composition, seasonal variability, and antifungal activity of Lavandula stoechas L. ssp. stoechas essential oils from stem/leaves and flowers. J Agric Food Chem;54(12):4364-70. Bhatnagar JK, Dunn MS. 1963. Histological studies of the genus Lavandula . III. Section Stoechas. linn., L. viridis L'herit.and L. pedunculata cav. Am J Pharm Sci Support Public Health ;35:288-306. Cavanagh HM, Wilkinson JM. 2002. Biological activities of lavender essential oil. Phytother Res;16(4):301-8. Christos N, Hassiotis. 2010. Chemical compounds and essential oil release through decomposition process from Lavandula stoechas in Mediterranean region. Biochemical Systematic and Ecology; 38(4): 493-501. Dadalioglu I, EvrendilekGA.2004. Chemical compositions and anti-bacterial effects of essential oils of Turkish oregano ( Origanum minutiflorum ), bay laurel ( Laurus nobilis ), Spanish lavender ( Lavandula stoechas L.), and fennel ( Foeniculum vulgare ) on common foodborne pathogens. J Agric Food Chem;52(26):8255-60. Díez Lázaro J, Kidd PS, Monterroso Martínez C. 2006. A phytogeochemical study of the Trás-os-Montes region (NE Portugal): possible species for plant-based soil remediation technologies. Sci Total Environ ; 354(2-3):265- 77. Freitas H, Prasad MN, Pratas J. 2004. Analysis of serpentinophytes from north-east ofPortugal for trace metal accumulation--relevance to the management of mine environment. Chemosphere;54(11):1625-42. Gabrieli C, Kokkalou E. 2003. A new acetylated glucoside of luteolin and two flavone glucosides from Lavandula stoechas ssp. stoechas.Pharmazie;58(6):426-7. Gámez MJ, Jiménez J, Risco S, Zarzuelo A. 1987. Hypoglycemic activity in various species of the genus Lavandula. Part 1: Lavandula stoechas L. and Lavandula multifida L. Pharmazie ;42(10): 706-7. Gilani AH, Aziz N, Khan MA, Shaheen F, Jabeen Q, Siddiqui BS, Herzig JW. 2000. Ethno-pharmacological evaluation of the anti-convulsant, sedative and anti-spasmodic activities of Lavandula stoechas L. J Ethnopharmacol;71(1-2): 161-7. Giray ES, Kirici S, Kaya DA, Türk M, Sönmez O, Inan M. 2007. Comparing the effect of sub-critical water extraction with conventional extraction methods on the chemical composition of Lavandula stoechas .Talanta. 15;74(4):930-5. Gören A, Topçu G, Bilsel G, Bilsel M, Aydo ğmu Z, Pezzuto JM. 2002. The chemical constituents and biological activity of essential oil of Lavandula stoechas ssp. stoechas. Z Naturforsch C;57(9-10):797-800. Gursoy UK, Gursoy M, Gursoy OV, Cakmakci L, Könönen E, Uitto VJ. 2009. Anti-biofilm properties of Satureja hortensis L. essential oil against periodontal pathogens. Anaerobe ;15(4):164-7. Khare CP. 2004. Indian herbal remedies: rational Western therapy, Ayurvedic, and other traditional usage botany. Pub by Springer-Verlag Berlin Heidelberg New York: 280-281. Kirmizibekmez H, Demirci B, Ye ilada E, Ba er KH, Demirci F. 2009. Chemical composition and anti-microbial activity of the essential oils of Lavandula stoechas L. ssp. stoechas growing wild in Turkey. Nat Prod Commun ;4(7):1001-6. Kokkalou E. 1998. The Constituents of the Essential Oil from Lavandula stoechas Growing Wild in Greece. Planta Med ;54(1): 58-9. Magro A, Carolino M, Bastos M, Mexia A. 2006. Efficacy of plant extracts against stored-products fungi. Rev Iberoam Micol;23(3):176-8. Manzoor-I-Khuda M, Khan MA. 1969. Isolation of new compounds from Lavandula stoechas . In: Said, M. (Ed.), Hamdard Pharmacopoeia. Hamdard National Foundation,Karachi: 480–482. Nadkarni KM. 1982. Indian Materia Medica, 3rd edition.Popular Prakashan, Bombay: 730. Nalda, NMJ, Bernal Yagüe JL, Diego Calva JC, Martín Gómez MT. 2005. Classifying honeys from the Soria Province of Spain via multivariate analysis. Anal Bioanal Chem;382(2):311-9.

310 Nogués S, Munné-Bosch S, Casadesús J, López-Carbonell M, Alegre L. 2001. Daily time course of whole-shoot gas exchange rates in two drought-exposed Mediterranean shrubs. Tree Physiol ;21(1):51-8. Roberts M, Green P. 2002. The essential Margaret Roberts: My 100 favorite herbs. 1st edition. Published by Spearhead. An imprint of New Africa Books (Pvt.) Ltd. Claremont: 63. Roller S, Ernest N, BuckleJ. 2009. The anti-microbial activity of high-necrodane and other lavender oils on methicillin-sensitive and resistant Staphylococcus aureus (MSSA and MRSA). J Altern Complement Med ;15(3):275-9. Sarac N, Ugur A. 2009. The in vitro antimicrobial activities of the essential oils of some Lamiaceae species from Turkey. J Med Food ;12(4):902-7. Sierra MJ, Millán R, Esteban E. 2009. Mercury uptake and distribution in Lavandula stoechas grown in soil from Almadén mining district (Spain). Food Chem Toxicol; 47(11):2761-7. Soylu EM, Soylu S, Kurt S. 2006. Antimicrobial activities of the essential oils of various plants against tomato late blight disease agent Phytophthora infestans .Mycopathologia;161(2):119-28. Sultan E, Kırıcı GS, Kaya DA, Türk M, Sönmez O, Đnan M.2008. Comparing the effect of sub-critical water extraction with conventional extraction methods on the chemical composition of Lavandula stoechas. Talanta; 74(4): 930–935. Topal U, Sasaki M, Goto M, Otles S. 2008. Chemical compositions and antioxidant properties of essential oils from nine species of Turkish plants obtained by supercritical carbon dioxide extraction and steam distillation. Int J Food Sci Nutr ;59(7-8):619-34. Topçu G, Ayral MN, Aydin A, Gören AC, Chai HB, Pezzuto JM. 2001. Triterpenoids of the roots of Lavandula stoechas ssp. stoechas. Pharmazie ;56(11):892-5. Traboulsi AF, Taoubi K, el-Haj S, Bessiere JM, Rammal S. 2002. Insecticidal properties of essential plant oils against the mosquito Culex pipiens molestus (Diptera: Culicidae). Pest Manag Sci.;58(5):491-5. Tzakou O, Bazos I, Yannitsaros A. 2009. Essential oil composition and enantiomeric distribution of fenchone and camphor of Lavandula cariensis and L. stoechas subsp. stoechas grown in Greece. Nat Prod Commun ; 4(8):1103-6. Ulubelen A, Goren N, Oleay Y. 1988. Longipinene derivativesfrom La vandula stoechas . Phytochemistry ; 27, 3966–3967. Ulubelen A, Olcay Y. 1989. Triterpenoids from La vandula stoechas .Fitoterapia ; 60: 475–476. Upson TM, J Grayer R, Greenham JR, A Williams C, Al-Ghamdi F, Chen F.2000. Leaf flavonoids as systematic characters in the genera Lavandula and Sabaudia . Biochem Syst Ecol. 1;28(10):991-1007. Uzun E, Sariyar G, Adsersen A, Karakoc B, Otük G, Oktayoglu E, Pirildar S. 2004. Traditional medicine in Sakarya province (Turkey) and antimicrobial activities of selected species. J Ethnopharmacol ;95(2-3):287-96. Vokou D, Chalkos D, Karamanlidou G, Yiangou M.2002. Activation of soil respiration and shift of the microbial population balance in soil as a response to Lavandula stoechas essential oil. J Chem Ecol ;28(4): 755-68. Yadav RK, Karamanoli K, Vokou D.2005. Bacterial colonization of the phyllosphere of mediterranean perennial species as influenced by leaf structural and chemical features. Microb Ecol ;50(2):185-96.

311