The Population Status of Lagochilus Setulosus Vved. and Its Biochemical Composition

Ecological Questions 32 (2021) 2: 111-118 http://dx.doi.org/10.12775/EQ.2021.018

The population status of Lagochilus setulosus Vved. and its biochemical composition

Arystanbek Eshibaev1, Zhanar Aimenova2,*, Lyazzat Akynova3, Lyazzat Nurseitova3, Aizhan Kopabaeva3

1ShymkentUniversity, Shymkent, 160031, Zhibekzholy street 131, Kazakhstan 2Muhtar Auezov South Kazakhstan University, Shymkent, 160012, Tauke Khan avenue 5, Kazakhstan 3South KazakhstanState Pedagogical University,Shymkent, 160012, Baitursinov street 13, Kazakhstan *corresponding author e-mail: [email protected] Received: 30 January 2021 / Accepted: 15 March 2021

Abstract. This article describes endemic species of the genus Lagochilus growing in the territory of South Kazakhstan – Lagochilus setulosus Vved. Its biochemical composition, namely qualitative and quantitative composition of biologically active compounds was investigated. The established composition of substances indicates that this plant species can be used in pharmaceutical biotechnology to create a biological preparation with a hemostatic effect based on Lagochilus setulosus.

Keywords: Lagochilus setulosus, South Kazakhstan, leaves and flowers, hemostatic agent, biological active compounds, deterpenes, lagochilin, lagochirzin.

1. Introduction tive compounds that will provide a long-term reliable raw material base for the pharmaceutical and cosmetology in- Wild flora of South Kazakhstan is represented by more dustries of Kazakhstan. Identification of the content of the than 3000 species of vascular plants. According to scien- main groups of important biologically active compounds tists, 25% of them are medicinal plants. Many species are in growing plant species will allow rational use of plant of practical value for the purposes of traditional and al- resources and will make it possible to significantly expand ternative medicine. However, the medicinal properties of the range of herbal medicinal raw materials. Some of these many of them still remain unexplored. plants are plants of the genus Lagochilus. There are more than 3000 plant species in the flora of Plants of the genus Lagochilus belong to the Lamiaceae South Kazakhstan, of which 553 are of interest as valu- family and are represented by 35 species distributed mainly able natural sources of medicinal substances (Flora of Ka- in Central Asia: in Kazakhstan, Iran, Afghanistan, Russia, zahstan, 1964). However, most of these plant species are Mongolia and China (Rehinger & Hedzh, 1982). Some still insufficiently known. Only a limited number of plant plants of this genus are used as hemostatic agents, for the species are used in the domestic pharmaceutical and cos- treatment of allergic dermatoses (Panossian & Wikman, metic industries. In this regard, the priority direction of 1982), and are also used as a medicine for clot retraction research is to identify potential sources of biologically ac- (Akopov, 1954) and glaucoma (Kadyroza, 1955). Among 112 Arystanbek Eshibaev, Zhanar Aimenova, Lyazzat Akynova, Lyazzat Nurseitova, Aizhan Kopabaeva

the various plant species of the genus Lagochilus, Lagochi- 2) The presence of tannins was determined by a specif- lus inebrians Bunge has long been used in folk medicine of ic gelatin deposition reaction (Sumina et al., 2006). the East as a medicinal plant due to its sedative properties 3) The presence of essential oil was determined by the (Perry et al., 2002). method described in State Pharmacopeia X (State However, according to Akopov I.E., Lagochilus setu- Pharmacopoeia of USSR, 1968). losus has the most pronounced stimulating effect on the 4) The presence of cardiac glycosides was determined blood clotting process (Akopov, 1981). However, this type by Keller-Kiliani Reaction (Syvkinet al., 1999). of plant is still insufficiently known and requires further 5) The presence of saponins was determined by the biological and chemical research. reaction to foaming (Ladygina et al., 1983). To solve this problem, we have already performed 6) The presence of phenolglycosides was determined studies on the macro-and micromorphological features of as follows (Petrov, 1978). Lagochilus setulosus (Aimenova et al., 2015a).As a result 7) The presence of iridoids was determined by the of the research, the anatomical structure of this plant was method described in State Pharmacopeia XI (State established using the method of scanning electron micros- Pharmacopoeia of USSR, 1989). copy. Gandular trichomes were found. 8) The presence of flavonoids was determined by cya- Also was performed a comparative research of the mac- nidin sample (Bandyukova, 1965). ro – and microelement composition of Lagochilus inebri- 9) The presence of coumarins was determined as fol- ans and Lagochilus setulosus (Aimenova et al., 2014). As lows: for the preparation of extraction from veg- a result of research, it was found that these plants differ in etable raw materials 2 g of crushed raw materials the quantitative content of macro-and microelements. Ac- (crushed flowers and leaves of plants), 20 ml of cording to the content of trace elements, the plant Lagochi- ethyl alcohol was poured and boiled for 15 min- lus inebrians surpasses Lagochilus setulosus, but the quan- utes with a reflux. After cooling, filtered. To 3-5 titative content of iron, magnesium, lithium, and aluminum ml of alcoholic extract was added 10 drops of 10% is 2-3 times higher in Lagochilus setulosus. At the same KOH in methanol and heated for 5 min in a water time, of all the identified macro – and microelements in bath (solution turned yellow), then added 5 drops of Lagochilus setulosus, as well as in Lagochilus inebrians, freshly prepared diazo reagent of Pauli Kutochku. the percentage content contains the most calcium, which Further, the solution acquired a cherry color, which in the presence of lagochirzin fully implements the process indicates the presence of coumarins. of plasma hemostasis. 10) The presence of anthracene derivatives was de- The next, necessary stage of the study of this plant spe- termined by the following method (Kurkin et al., cies is the study of its distribution area, as well as the study 2016). of its biological composition. 11) The presence of sugars was determined by the method (Fialkov, 1946). 12) The presence of alkaloids was determined by the 2. Objects and methods method (Ladygina, 1983). 13) The presence of resinous substances was deter- For the research, the flowers and leaves of Lagochilus setu- mined by the decrease in the mass of the sample losus were collected during the mass flowering period in after exhaustive extraction in the soxlet apparatus July. To do this, the tops of the stems of plants 20-30 cm with a capacity of 1 l for 20 h and subsequent dry- long were cut off, leaving 1-2 plants per 1m2 for seed- ing to a constant weight at a temperature of 105°C. ing and population renewal. Drying was carried out in the Further, the method of fractionation using chloro- shade in a well-ventilated room, laying it out in a thin layer form and acetone solvents was used to separate res- on a tarpaulin. Then, after drying, the stems were threshed inous substances (Kuznetsov et al., 2004). with a wooden stick, separated and discarded. Then the Quantitative determination of the content of the main yellowed and browned parts were removed and the raw groups of biologically active substances of Lagochilus materials were crushed in a mechanical shredder. Thus, setulosus performed according to the following methods: the finished raw material contained flowers and leaves that 1) essential oil by the method of (State Pharmacopoeia of retained their natural color. the USSR, 1968). Qualitative determination of the content of the main 2) resinous substances by the method: the leaves and flow- groups of biologically active substances in the phytomass ers of Lagochilus setulosus crushed to a particle size of of Lagochilus setulosus was performed according to the 1-2 mm and subjected to extraction of non-polar sol- following methods: vent, hexanal to conventional Soxhlet extractions for 1) The presence of diterpenes was determined by paper the separation of resinous substances. The yield of res- chromatography (Belenkij et al., 1983). inous substances depending on the terms of raw mate- V2 – the volume of ethanol extract was taken for use on chromatogram, ml; V2 – the volume of ethanol extract was taken for use on chromatogram, ml; V3 – the volume of eluate; The population status of Lagochilus setulosus Vved.V2 – and the itsvolume biochemical of ethanol composition extract was taken for use 113on chromatogram, ml; V3 – the volume of eluate; D363 – optical density of solution at λ = 363 nm; V3 – the volume of eluate; D – optical density of solution at λ = 363 nm; D1%/1cm363 – the rate of absorption of rutinat λ = 363 nm (268.4); D363 – optical density of solution at λ = 363 nm; rial preparation and storage was 6-9% of the weight of D1%/1cmD1%/1cm – the – therate rate of ofabsorption absorption of of rutinat rutinat λ λ= = 363 363 nmnm (268.4); m – Dthe1%/1cm weight – of the the rate portion of absorption of the raw of material, rutinat λ g=; 363 nm (268.4); the absolutely dry residue. m – the weight(268.4); of the portion of the raw material, g; 3) diterpene1) essentiallagochilin oil according by the method to the of method: (State Pharmacopoeia 10 g of mw – of mlossthe the – theweightin USSR weight weight ,of 1968 of theof raw )theportion. portionmaterials of theof during the raw raw material, drying, material, %; g; g ; finely powdered raw material (flowers and leaves of w – wloss – loss in weightin weight of rawof raw materials materials during during drying, drying, %; %; 2) resinous substances by the method: the leaves 0.and067w flowers– lossconversion in of weight Lagochilus factor of raw to 20 materialssetulosus ml of extract; during drying, %; Lagochilus setulosus was placed in a 250 ml flask and 0.0670.067 – conversion – conversion factor factor to 20to 20ml mlof ofextract; extract; l – layer thickness, cm. 100crushed ml chloroform to a particle was size poured. of 1-2 The mm flask and subjectedwith the con to -extractionl – layer0.067 of thickness, non– conversion-polar cm.solvent, factor hexanal to 20 ml to of extract; l – layer thickness, cm. tentsconventional was attached Soxhlet to theextractions reflux and for heatedthe separation in a water of resinous l –substances.6 layer) iridoids thickness, Theby the yield cm following. of resinous technique: the content of the sum of iridoids in terms of 6) iridoids by the following technique: the content of the sum of iridoids in terms of bath for 1 hour. The extract was then cooled and fil- 6)harpagid iridoids and by absolutely the following dry rawtechnique: materials the (%) content was calculated of the by the formula: tered.substances In the depending same way onchloroform the terms extraction of raw material was re -preparationsum of and 6iridoids) iridoidsstorage in termswasby the 6 of- 9%following harpagid of the andtechnique: absolutely the drycontent of the sum of iridoids in terms of harpagid and absolutely dry raw materials (%) was calculated by the formula: peatedweight 5-6 of thetimes absolutely (from one dry rawresidue. material). Chloroform rawharpagid materials and (%)absolutely was calculated dry raw materialsby the formula: (%) was calculated by the formula: extracts were combined and chloroform distilled to pro- = , (2) 3) diterpene lagochilin according to the method: 10 g of finely powdered raw material duce a dry residue. 10 ml of distilled water was added = , ( (2)2)

to(flowers the dry and residue leaves and of heatedLagochilus (5 min) setulosus in a water was placed bath, in a 250 ml flask and 100 ml chloroform= , (2) where: adding 15 ml of 10% NaOH and heated in a water bath was poured. The flask with the contents was attached to the refluxwhere and: heated in a water bath for for 30 min. The water-alkaline mixture, after cooling, where:А – whereoptical: density of the tested solution; was1 hour. repeatedly The extract treated was with the ethyln cooled ether and(5-6 filtered.times). TheIn theА same –А optical – way optical densitychloroform density of theof extraction the tested tested solution; was solution ; m – Аraw – opticalmaterial density linkage, of theg; tested solution; combinedrepeated 5essential-6 times (fromextracts one were raw material).concentrated Chloroform to 10 extractsm –m raw – wereraw material materialcombined linkage, linkage, and g; chloroform g; ml and left for the crystallization of lagochilin, which W – humidity ofof rawraw materialsmaterials;; distilled to produce a dry residue. 10 ml of distilled water mwas – rawadded material to the linkage, dry residue g; and was separated by filtration through a suspended filter, 5. 56W – – the humidity rate of ofabsorption raw materials of the; reaction products with 5. 56W – –the humidity rate of absorptionof raw materials of the; reaction products with hydroxylamine and iron (III) chloride. driedheat edand (5 weighed. min) in Lagochilina water bath, recrystallization adding 15 ml wasof 10% per- NaOH andhydroxylamine heated in a water and bathiron (III)for 30 chloride. 5. 756) coumarins– the rate ofaccording absorption to theof the technique reaction. Theproducts optical with density hydroxylamine of the solutions and iron was (III) measured chloride. formed with acetone. 5. 56 – the rate of absorption of the reaction products with hydroxylamine and iron (III) chloride. min. The water-alkaline mixture, after cooling, was repeatedly treated7) coumarins with ethylaccording ether to (5 the-6 technique. The optical density of the solutions was measured 4) diterpene lagochirzin by the method of water-alkaline 7)at thecoumarins following according wavelengths: to the isopimpinellintechnique. The –optical 313 nm, den bergapten- – 311 nm. The percentage of times). The combined essential extracts were concentrated to7 ) 10coumarins ml and according left for to thethe technique. The optical density of the solutions was measured solution, after removal of lagochilin neutralized with sityizopimpinellin atof the the following solutions and bergapten wavelengths:was measured was isopimpinellincalculated at the following by the – 313 formula:wave nm,- bergapten – 311 nm. The percentage of 20% H SO solution until slightly acid reaction (pH=5) lengths:at the isopimpinellin following wavelengths: – 313 nm, bergaptenisopimpinellin – 311 –nm. 313 The nm, bergapten – 311 nm. The percentage of crystallization2 4 of lagochilin, which was separated by filtrationizopimpinellin through a suspended and bergapten filter, wasdried calculated by the formula: and the mixture was repeatedly treated with chloroform percentage of izopimpinellin and bergapten was calculated and weighed. Lagochilin recrystallization was performed with izopimpinellinacetone. and bergapten was calculated by the formula: (5-6 times). Chloroform extract was combined, concen- by the formula: , (3) x= trated and4) diterpenechloroform lago hadc hirzintetroxide by in the distilled. method The of dry water -alkalinex= solution, after, removal of (3) residue was obtained in an amount of 0.22 g. It was x= , (3(3)) lagochilin neutralized with 20% H2SO4 solution until slightly acid reaction (pH=5) and the passed through a column with 10 g of silica gel (column where:

diametermixture 2was cm, repeatedly height 20 treatedcm) and with eluted chloroform with a mixture (5-6 times). whereChloroform: extract was combined, V1 –where extraction: volume, ml; ofconcentrated ethyl ether andand petroleumchloroform ether had (40:1). tetroxide A total in distilled.of 20 where:TheV 1 dry– extraction residue wasvolume, obtained ml; in an V2 – volume of extraction applied to the chromatogram, ml; fractions (5 ml) were obtained; 5-12 fractions contain- V1 –V extraction1 – extraction volume, volume, ml; ml; amount of 0.22 g. It was passed through a column with 10 g ofV 2silica – volume gel (column of extraction diameter applied 2 cm, to the chromatogram, ml; ing lagochirzin (chromatography on plates “Sorbfil”) V3 –– volumethe volume of extraction of eluate, applied ml; to the chromatogram, ml; 2 V2 – volume of extraction applied to the chromatogram, ml; were combined, evaporated, the dry residue was dried V –V the3 – thevolume volume of eluate,of eluate, ml; ml ; height 20 cm) and eluted with a mixture of ethyl ether andm 3 petroleum– the weight ether of the(40:1) portion. A total of the of raw20 material, g; and weighed. m – Vthe3 – weightthe volume of the of portion eluate, ofml ;the raw material, g; fractions (5 ml) were obtained; 5–12 fractions containing lagomc hirzin– the weight(chromatography of the portion on ofplates the raw material, g; 5) flavonoids by the following technique: the optical den- m – thespecific weight absorptionabsorption of the portion raterate:: isopimpinellin ofisopimpinellin-518, the raw material,-518, bergapteng ber; - -651; sity"Sorbfil") of eluates were was combined, measured evaporated, on a spectrophotometer the dry residue was dried andgapten-651; – weighed. specific absorption rate: isopimpinellin-518, bergapten-651; – specific absorption rate: isopimpinellin-518, bergapten-651; in a cuvette5) flavonoids with a layer by the thickness following of technique:1 cm at a thewave optical- D density2 – the optical of eluates density was ofof measured thethe eluate;eluate; on a length of 363 nm against the background of the eluate w – Dloss– the in weightoptical densityof raw materialsof the eluate; during drying, %. w – loss2 in weight of raw materials during drying, %. ofspectrophotometer the idle experiment. in aThe cuvette percentage with a oflayer routine thickness in raw of 1 cm Dat2 –a thewavelength optical density of 363 of nm the against eluate; w – loss in weight of raw materials during drying, %. materialsthe background in terms of ofthe absolutely eluate of thedry idle raw experiment. materials was The percentage8) phenolglycosidesw 8–) lossphenolglycosides of inroutine weight inby ofraw the raw by materials following materialsthe following in technique:during technique: drying, 1 ml %.1 ofml of 0.1 N. iodine solution corresponds calculated by the formula: 0.1 N. 8iodine) phenolglycosides solution corresponds by the following to 0.01361 technique: arbutin. 1 ml of 0.1 N. iodine solution corresponds terms of absolutely dry raw materials was calculated by theto formula: 0.013618 )arbutin. phenolglycosides The percentage by the of following arbutin in technique: plant material 1 ml ofx in0.1 terms N. iodine of absolutely solution correspondsdry raw Theto 0.01361 percentage arbutin. of arbutin The percentage in plant material of arbutin x in termsplant material x in terms of absolutely dry raw materialsofto absolutely 0.01361 was calculated arbutin. dry raw The by materials thepercentage formula: was of calculated arbutin in by plant the material x in terms of absolutely dry raw (1) formula:materials was calculated by the formula: 5 (1) materials was calculated by the formula: 5 5 where: , (4)(4) where: K1 – the ratio of elution (1.195);

V1 –K volume1 – the ratio of ethanol of elution extract (1.195); after dissolution of dry residue; ml; where:where: V1 – volume of ethanol extract after dissolution of dry residue; ml; V2 – the volume of ethanol extract was taken for use on V – Vvolume – volume of 0.1of 0.1N. N.iodine iodine solution solution consumed consumed for for titra titration,- ml; chromatogram, ml; tion, ml; 4 m – the weight of the portion of the raw material, g; V3 – the volume of eluate; m – the weight of the portion of the raw material, g;

D363 – optical density of solution at λ = 363 nm; w – wloss – lossin weight in weight of raw of raw materials materials during during drying, drying, %. %. 9) alkaloids by the following technique: The percentage in terms of absolutely dry raw materials x was calculated by the formula:

, (5)

where: V – the volume of 0.02 n NaOH used for titration, ml; m – the weight of the sample of raw materials corresponding to the volume of ether extraction, g; w – loss in weight of raw materials during drying, %. 10) sugars by the method of Willstatter and Sudle. The optical density of the studied solution was recorded on a spectrophotometer at a wavelength λ = 582 nm. The sugar content in the sample was determined by the calibration curve based on glucose. To do this, 50 ml of a solution containing 10 mg/ml of glucose was prepared, and then the remaining solutions were obtained by dilution according to Table 1.

Table 1. Method of dilution of the solution to obtain a glucose calibration curve

Amount of initial glucose Amount of water, Number Content of glucose, mg/ml solution, ml ml 1 0.5 0.5 9.5 2 1.0 1.0 9.0 3 2.5 2.5 7.5 4 5.0 5.0 5.0 5 7.5 7.5 2.5 6 10.0 10.0 0

The number of reducing sugars (x) was calculated by the formula:

, (6)

where: с – sugar content in the sample, found on the calibration curve; V – the volume of the extract obtained from the sample; m – weight of the sample in grams.

, (4)

where: V – volume of 0.1 N. iodine solution consumed for titration, ml; 114 Arystanbek Eshibaev, Zhanar Aimenova, Lyazzat Akynova, Lyazzat Nurseitova, Aizhan Kopabaeva m – the weight of the portion of the raw material, g; w – loss in weight of raw materials, during drying, %. (4 ) 9) alkaloids9) byalkaloids the following by the technique:following Thetechnique: percentage The inpercentage hour. The in extractterms of was absolutely then cooled dry and raw filtered. In the same terms of absolutely dry raw materials x was calculated way chloroform extraction was repeated 5-6 times (from materialswhere: x was calculated by the formula: by the formula: one raw material). Chloroform extracts were combined and V – volume of 0.1 N. iodine solution consumed for titration,chloroform ml; distilled to produce a dry residue. m – the weight of the portion of, the raw material, g; (5)( 5) To the dry residue, 10 ml of distilled water was added and heated (5 min) in a water bath, then 15 ml of 10% w – loss in weight of raw materials during drying, %. where: NaOH was added and heated in a water bath for 30 min. V – wherethe volume:9 ) alkaloids of 0.02 by n NaOHthe following used for technique: titration, ml;The percentageThe water-alkaline in terms of absolutelymixture, after dry cooling,raw was repeated- m –V materialsthe – theweight volume x wasof the ofcalculated sample0.02 n NaOH of by raw the used materialsformula: for titration, correspond ml; - ly treated with ethyl ether (5-6 times). The combined es- ing to the volume of ether extraction, g; sential extracts were concentrated to 10 ml and left for w –m loss – the in weight ofof theraw sample materials of rawduring materials drying, corresponding %. the tocrystallization the volume of of ether lagochilin, extraction, which g; was separated by w – loss in weight of raw materials, during drying, %. ( 5) filtration through a suspended filter, dried and weighed. 10) sugars by the method of Willstatter and Sudle. The Lagochilin recrystallization was performed with acetone. 10) sugars by the method of Willstatter and Sudle. The optical density of the studied optical density of the studied solution was recorded The aqueous-alkaline solution, after removal of lagochi- where: solutionon a spectrophotometer was recorded on at a spectrophotometera wavelength λ = 582at a nm.wavelength lin was λ =neutralized 582 nm. The with sugar 20% content H2SO4 insolution to a slightly theVThe – samplethe sugar volume content was ofdetermin 0.02in the n sampleNaOHed by thewasused calibration determined for titration, curve by ml; the based acidic on glucose.reaction To(pH=5) do this, and 50 the ml mixture of a was repeatedly calibration curve based on glucose. To do this, 50 ml treated with chloroform (5-6 times). Chloroform extract m – the weight of the sample of raw materials corresponding to the volume of ether extraction, g; solutionof a solution containing containing 10 mg/ml 10 mg/ml of glucose of glucose was was prepared, pre- andwas thencombined, the remaining concentrated solutions and chloroformwere had tetroxide obtainedwpared, – loss and inby weightthen dilution the of remainingaccording raw materials solutionsto T ableduring 1 were. drying, obtained %. in distilled. The dry residue was obtained in an amount of by dilution10) sugarsaccording by tothe Table method 1. of Willstatter and Sudle.0.22 The g. the optical Dry residuedensity was of thepassed studied through a column with 10 g of silica gel (column diameter 2 cm, height 20 cm) Table 1. Method of dilution of the solution to obtain a glucose calibration curve solution was recorded on a spectrophotometer at a wavelengthand eluted λ = 582 with nm. a Themixture sugar of content ethyl ether-petroleum in ether Table 1. Method of dilution of the solution to obtain a glucose (40:1); 20 fractions (5 ml) were obtained. 5-12 fractions the samplecalibration was curve determined by the calibrationAmount curve ofbased initial on glucoseglucose. To Adomount this, 50of waterml of, a Number Content of glucose, mg/ml contained lagochirzin (chromatography on plates “Sorb- solution containing 10 mg/ml of glucose was prepared,solution, and mlthen the remaining solutionsml were Content Amount of initial fil”), which were combined, evaporated, the dry residue 1 0.5 Amount 0.5 9.5 Numberobtained ofby glucose, dilution accordingglucose solution,to Table 1. was dried and weighed. 2 1.0 of water, ml 1.0 9.0 3 mg/ml 2.5 ml 2.5 7.5 Table1 4 1. Method0.5 of dilution5.0 of 0.5 the solution to obta9.5 in a glucose5.0 calibration curve 5.0 5 7.5 7.5 3. Results and2.5 discussion 2 6 1.0 10.0 1.0 A9.0mount of10 initial.0 glucose Amount0 of water, Number Content of glucose, mg/ml 3 2.5 2.5 7.5 solution,The mlresults of the taxonomicml analysis established the dis- 1The number of reducing0.5 sugars (x) was calculated by0tribution. 5the formula: areas of plants of9 the.5 genus Lagochilus in three 4 2 5.0 1.0 5.0 5.0 1floristic.0 regions of the Turkestan9.0 region (Aimenova et al., 5 3 7.5 , 2 . 5 7.5 2.5 (6) 22015b)..5 7.5 4 5.0 5.0 At the same time, Lagochilus5.0 setulosus grows in the

6 5 10.0 7.5 10.0 0 7soil.5 and climatic conditions2.5 of the foothill zone of the where: 6 10.0 10western.0 Tien Shan, which 0is characterized by a moder- Theс – sugar number content of reducing in the sample, sugars found(x) was on calculatedthe calibration by curve;ate temperature regime, moisture availability and typical the formula:The number of reducing sugars (x) was calculated byserozem. the formula: V – the volume of the extract obtained from the sample; m – weight of the sample in grams. Morphological features of Lagochilus setulosus are as- , 6 (6 )(6) sociated with the conditions of the place of growth of this species, which determines its characteristic macro- and where:where: micromorphological features. These features include the с – sugar content in the sample, found on the calibration modification of the young leaves of the plant into spines, сcurve; – sugar content in the sample, found on the calibration curve;a reduction in the size of the leaves, the growth of sharp V – Vthe – thevolume volume of ofthe the extract extract obtained obtained from from the the sample; sample; mspines – weight at the of theend sample of the leavesin grams. and sepals. The leaf epider- m – weight of the sample in grams. mis is covered with a thick layer of cuticle, the mesophyll 6 consists of 4 compressed layers of palisade tissue. The veg- For isolating diterpene lagochirzin of phytomass etative and generative organs are covered with glandular Lagochilus setulosus we used the following method: 10 g trichomes that produce a specific sticky substance around of finely ground raw materials (flowers and leaves of the seeds that keeps them moist. Lagochilus setulosus) was placed in a 250 ml flask and Lagochilus setulosus is a semi-shrub, reaching a height 100 ml chloroform was poured. The flask with the contents of 20-60 cm. The root of the plant stem, the stems at the was attached to the reflux and heated in a water bath for 1 base stiffening, thin, glabrous or sparsely and protruding The population status of Lagochilus setulosus Vved. and its biochemical composition 115

Lagochilus setulosus is a semi-shrub, reaching a height of 20-60 cm. The root of the plant stem,bristly, thelater stems white, shiny,at the 30-80 base cm stiffening, tall; leaves rhombicthin, glabrous in As or it sparselycan be seen and from protruding Table 2, in bristly, the phytomass later of outline, broadly ovate. Lagochilus setulosus were identified 9 specific qualitative white,We shiny,found four 30- populations80 cm tall; of leaves Lagochilus rhombic setulosus in outline, in reactions broadly to the ovate. presence of biologically active substances, the Kazygurt district (Turkistan Region in Southern Ka- which are important organic compounds used in pharma- zakhstan,We whose found administrative four populations center is theof villageLagochilus of Ka- setulosusceuticals, perfumeryin the Kazygurt and medicine. district However, (Turkistan among these Regionzygurt). The in Southern patches of Kazakhstan,plants vary from whose 1.3 to administrative 4.5 ha. substances center we ishave the not village found tannins,of Kazygurt) anthracenes,. The sapo - However,Lagochilus the density setulosus of plants is ina semithe area-shrub, (coordinates reaching nins a height and cardiac of 20 glycosides.-60 cm. The Qualitative root of reactions the plant to these stem,patches41.759°N the of stems69.41°E) plants at vary isthe not frombase high 1.3stiffening,and toit 4.5does ha. notthin, However, exceed glabrous compounds the or density sparsely showed of plantsand negative protruding in theresults. area bristly, (coordinates later 2 1.2±0.1 plants per m (Fig. 1). In the next stage of our 2research, the presence of previ- white,41.759°NThe shiny, projective 69.41°E) 30-80 cover cm is of nottall; the high leavessoil andwith rhombic itvegetation does notin isoutline, exceed no ously broadly1.2±0.1 identified ovate.plants biologically per m (Fig.active 1).substances in the phy- more than 15.3±1.3%. Plants are found in individual speci- tomass of plants was tested in experiments with thin-layer mens,We bushy found shape, four height populations 35-40±1.7 cmof inLagochilus accordance setulosuspaper chromatography. in the Kazygurt As studies district have shown, (Turkistan all qualita - Regionwith Figure in Southern2 A, B. Kazakhstan, whose administrativetive reactions center is were the reliable, village all of of Kazygurt) the biologically. The active The results of qualitative analyses of biological active substances listed in table 3 were identified by comparing patchessubstances of plantspresence vary are givenfrom in 1.3 Table to 4.52. ha. However, the standarddensity samplesof plants – «witnesses». in the area (coordinates 41.759°N 69.41°E) is not high and it does not exceed 1.2±0.1 plants per m2 (Fig. 1).

Figure 1. The total area of the populations of Lagochilus setulosus in Kazygurt district

The projective cover of the soil with vegetation is no more than 15.3±1.3%. Plants are

found in individual specimens, bushy shape, height 35-40±1.7 cm in accordance with Figure 2 A,Figure B. 1. The total area of the populations of Lagochilus setulosus in Kazygurt district Figure 1. The total area of the populations of Lagochilus setulosus in Kazygurt district

The projective cover of the soil with vegetation is no more than 15.3±1.3%. Plants are found in individual specimens, bushy shape, height 35-40±1.7 cm in accordance with Figure 2 A, B.

А) general view of the plant; B) type of flowering plant shoot А) general view of the plant; B) type of flowering plant shoot Figure 2. Appearance of Lagochilus setulosus plants Figure 2. Appearance of Lagochilus setulosus plants

8 А) general view of the plant; B) type of flowering plant shoot

Figure 2. Appearance of Lagochilus setulosus plants

116 Arystanbek Eshibaev, Zhanar Aimenova, Lyazzat Akynova, Lyazzat Nurseitova, Aizhan Kopabaeva

Table 2. Results of preliminary phytochemical screen- was previously synthesized from lagochilin as a result of ing of flowers and leaves of Lagochilus setu- multi-stage reactions, was performed by Zainutdinov on losus L. inebrians. L. setulosus is one of the rare plant species of Qualitative this genus that contains lagochirzin in its pure form. This Number Biologically active substance reaction fact suggests a higher hemostatic activity of L. setulosus in comparison with L. gypsaceus (Zainutdinov, 1993). 1 tannins not identified

2 phenolic glycosides Identified Table 3. Results of the study of the quantitative content of the 3 anthracenediones not identified main groups of biologically active substances in the biomass of leaves and flowers of Lagochilus setulosus 4 sugars Identified plants 5 saponines not identified Biologically active Quantitative content in % Number 6 essential oil Identified substance to weight of sample 7 cardiac glycosides not identified 1 Essential oil 0.12 ± 0.004 8 flavonoids Identified Resinous 2 8.45 ± 0.250 9 coumarins Identified substances 10 resinous substance Identified Diterpenes 3 Lagochilin 0.75 ± 0.005 11 diterpenes Identified Lagochirzin 1.68 ± 0.023 12 iridoids Identified 4 Flavonoids 0.35 ± 0.002 13 alkaloids identified 5 Iridoids 0.81± 0.014 6 Coumarins 0.11± 0.001 As shown by the results of quantitative studies in the 7 Phenolic glycosides 2.75±0.011 phytomass of plants Lagochilus setulosus maximum meas- 8 Alkaloids 0.70±0.002 ure have sugar (of 8.81%), resinous substances (of 8.45%), phenolic glycosides (2.75%) and diterpenes (2.43%). The 9 Sugars (in total) 8.81± 0.044 content of the remaining compounds is below one percent – 0.11-0.81% (Table 3). The established quantitative indicators of biologically Phenolic glycosides, which were also found in L. setu- active substances characterize the high pharmaceutical val- losus, suggest the antioxidant activity of this plant. Since ue of raw materials of plants of this species. Since these the literature data provides a clear link between the strong substances are usually synthesized in plants in small quan- correlation between the total phenol content and the anti- tities, although they play a very important role in the me- oxidant (DPPH, CUPRAC, and FRAP) properties of the tabolism of the body. The obtained quantitative indicators plant of the genus Lagochilus (Akramov et al., 2019). of biological active substances in plants of Lagochilus set- In the study of Ebrahimi et al. (2020) also were inves- ulosus consistent with the results of other authors’ research. tigated the total of 17 medicinal plants with hemostatic In the article of Akramov et al. (2019) is shown com- activity. The most frequently studied plant families were parative study on the chemical composition and biological Compositae, Lamiaceae, Fabaceae, and Asteraceae. The activities of the essential oils of three Lagochilus species majority of the plants were prepared in the form of aqueous collected from Uzbekistan. There are L. gypsaceus, L. ine- or organic extracts of leaf, rhizome, flower, bark, pollen brians and L. setulosus. L. gypsaceus was chosen by him to or the whole plant, and for some plants the active com- the study its phytochemical composition, which should be ponents were isolated. As a result, it was shown that the related to its most relevant biological properties. Studies of hemostatic activity of plant extracts is mainly attributed L. gypsaceus have revealed iridoid glycosides, diterpenes, to several mechanisms, including coagulation stimulation flavonoids and sterols. Regarding diterpenoids – which, ac- via increasing the factor XII activity and plasma fibrino- cording to the conducted studies, determine the hemostatic gen levels, the fibrinolysis inhibition, vascular or smooth function of plants of the genus Lagoсhilus, in L. gypsaceus muscle constriction and platelet aggregation. Ageratum co- only the diterpene lagochilin was found, while lagochirzin nyzoides and Typha latifolia are the plants with the most in its pure form is present in L. setulosus which grows in vivo and in vitro evidence of hemostatic activity. It is in the territory of South Kazakhstan. Lagochirzin, which propounded that the bioactive compounds which are often The population status of Lagochilus setulosus Vved. and its biochemical composition 117

involved in the bleeding control are categorized as tan- Uzbekistan. Natural Product Research 2019. DOI: nins, saponins, glycosides and other phenolics. Moreo- 10.1080/14786419.2019.1655417. ver, the anti-fibrinolytic effect of browplasminin (tannin), Akramov D.Kh., Bacher M., Böhmdorfer S., Rosenau 8-O-acetyl shanzhiside methylester (iridoid glycoside) and T., Zengin G., Potthast A., Nahar L., Sarker S.D. & lignin were confirmed. Other isolated hemostatic com- Mamadalieva N.Z., 2020, Phytochemical analysis and pounds include glycoconjugate from Lythrum salicaria, biological evaluation of Lagochilus species from Uz- saponins from Panax notoginseng, and Gallic acid, vanil- bekistan. Industrial Crops & Products 2020. https://doi. lic acid and luteolin from Sedum aizoon. org/10.1016/j.indcrop.2020.112715. Bandyukova V.А., 1965, The use of color reactions for the detection of flavonoids through chromatography on pa- 4. Conclusion per. Plant Resources 1(4): 591-597 [in Russian]. Belenkij B.G., Volynec M.P., Gankina E.S., 1983, Modern Thus, the distribution areas of plants of the genus Lagochi- thin-layer chromatography. Journal of the All-Union lus were established in three floristic regions of the Turke- Chemical Society 28(1): 30-34 [in Russian]. stan region. Qualitative and quantitative analyses of the Ebrahimi F., Torbati M., Mahmoudi J., & Valizadeh given biomass were carried out. The established composi- H., 2020, Medicinal Plants as Potential Hemostatic tion of substances characterizes this type of plant as prom- Agents. Journal of Pharmacy and Pharmaceutical Sci- ising for use in pharmaceutical biotechnology in the crea- ences 23(1): 10-23. tion of a biological drug with a hemostatic effect based on Fialkov Ya.А., 1946, Research methods of medicinal sub- Lagochilus setulosus. stances. Medgiz, Moscow, 255 pp. [in Russian]. Flora of Kazakhstan, 1964, Vol. VII. Nauka,Alma-Ata, 429 pp. [in Russian]. References Kadyroza K.K., 1955, Lagochilusin the treatment of glaucoma. Bulletin of Ophthalmology 34: 32-35 [in Rus- Aimenova Zh.E., Eshibaev A.A. & Zaynutdinov U.N., sian]. 2015b, Research of areas of distribution of valuable Kurkin V.А., ShmygarevaА.А. &SańkovА.N., 2016, De- medicinal species of plants of Lagochilus Bunge (Lami- velopment of new approaches to standardization of aceae) genus of South Kazakhstan Territory. The fourth «Trostinka» collection. Scientific Statements of Bel- European conference on Biology and Medical sciences, gorod State University, Series: Medicine. Pharmacy 13th January 2015,Vienna, p. 4-9. 33(5), 226 pp. [in Russian]. Aimenova Zh.E., Eshibaev А.А., & Akynova L.А., 2015a, Kuznetsov B.N., Kuznetsova S.А. & TarabańkoВ.E., 2004, Research of macro- and micromorphological features New methods of production of chemical products from of Lagochilus setulosus growing in the South Kazakh- biomass of Siberian trees. Russian Chemical Journal stan region. Bulletin of the L.N.Gumilyov Eurasian Na- 48(3): 4-19 [in Russian]. tional University 4(107): 126-132 [in Russian]. Ladygina E.A., 1983, Chemical analysis of medicinal Aimenova Zh.E., Zainutdinov U.N. & Matchanov A.D, plants. High School Publishing House, Moscow, 152 2014, Active ingredients of the plant Lagochilus in- pp. [in Russian]. ebrians. Collection of materials of the international Ladygina Е.Ya., Safronich L.N. & Otryashenkova V.E., scientific and practical conference: Pharmaceutical ed- 1983, Chemical analysis of medicinal plants: textbook ucation, science and production-a reference point for for pharmaceutical universities. High School Publish- the strategy “Kazakhstan-2020”, 23-24 October 2014. ing House, Moscow, 225 pp. [in Russian]. Conference Book No. 3(68), Vol II, p. 103-106, Shym- Panossian A, Wikman G., 2008, Pharmacology of Schisan- kent, Republic of Kazakhstan [in Russian]. dra chinensis Bail.: an overview of Russian research Akopov I.E., 1954, The effect of Lagochilus preparations and uses in medicine. J. Ethnopharmacol. 118(2): 183- on blood clotting. Bulletin of Experimental Biological 212. DOI: 10.1016/j.jep.2008.04.020. E-pub 2008 April Medicine 37: 49-54 [in Russian]. 24, PMID: 18515024. Akopov I.E., 1981, Hemostatic plants. “Medicine” UzSSR, Perry E.K., Ashton H. & Young A.H. (eds.), 2002, Neu- Tashken, p.125-127 [in Russian]. rochemistry of consciousness: neurotransmitters in the Akramov D.Kh., Zengin G., Kang S.Ch., Tojibaev K.Sh., brain. John Benjamins Publishing Company, Amster- Mahomoodally M.F., Azimova S.S. & Mamadalieva dam. e-Book ISBN: 9789027297891. N.Z., 2019, Comparative study on the chemical Petrov K.P., 1978, Methods of biochemistry of plant prod- composition and biological activities of the essen- ucts. Higher School Publishing House, Kiev, 154 pp. tial oils of three Lagochilus species collected from [in Russian]. 118 Arystanbek Eshibaev, Zhanar Aimenova, Lyazzat Akynova, Lyazzat Nurseitova, Aizhan Kopabaeva

Rehinger K.H. & Hedzh I.K., 1982, Flora of Iran, Labia- cation. Manual. N.G. Chernyshevsky Saratov National tae. Academic Reference Book, Grac [in Russian]. Research University, Saratov, 110 pp. State Pharmacopoeia of USSR, 1968, General methods of Syvkin А.I., Selemenev V.F. & Suhoverhova Е.А., 1999, analysis. Medicine Publishing House, Series 10, Health Physico-chemical and biological methods of drug eval- Ministry of USSR, Moscow, 818pp. [in Russian]. uation: textbook.Publishing House of Voronezh Uni- State Pharmacopoeia of USSR, 1989, General methods of versity, Voronezh, 368 pp. [in Russian]. analysis. Medicine Publishing House, Series 11, Health Zainutdinov U.N., 1993, Diterpenes of Lagochilus genus Ministry of USSR, Moscow, 400 pp. [in Russian]. plants. Dissertation for the degree of Doctor of Chemis- Sumina E.G., Shtykov S.Н.&Turina N.V., 2006, Thin-layer try, 02.00.10, Tashkent State University, Tashkent, 339 chromatography. Theoretical bases and practical appli- pp. [in Russian].