Chapter - 3 PHARMACOGNOSY OF TEPHROSIA PURPUREA AND SYZYGIUM CUMINII

Transcription

1 Chapter - 3 PHARMACOGNOSY OF TEPHROSIA PURPUREA AND SYZYGIUM CUMINII

2 Tephrosia purpurea Pers. Physical evaluation Aerial parts of Tephrosia purpurea were collected from the waste lands around Vallabh Vidyanagar in the month of March. The macroscopic studies were carried out with the help of naked eyes and simple hand lens. For anatomical studies, hand sections were cut out, cleaned, stained, mounted and observed under microscope to confirm the identity of the drug. Voucher of the specimen was deposited in the Department of Pharmacognosy, A. R. College of Pharmacy, Vallabh Vidyanagar, Gujarat. Macroscopic features Tephrosia purpurea Pers. (Fig. 3.1) is found to be a polymorphic, much branched, sub erect, perennial herb, cm high. Leaves were imparipinnate, 7-15 cm. Long: leaflets 8-12 mm. Narrow, oblanceolate, glabrous above, obscurely silky below; flowers purple, in leaf-opposed recemes; pods slightly recurved, cm x 4.5 mm., glabrescent; seeds 6-11, greenish gray and smooth. 62

3 Fig. 3.1: External morphology of Tephrosiapurpurea Microscopy of root The young root (Fig. 3.2A and 3.2B) shows a single layered epiblema composed of the walled narrow, cubical to tangentially elongated cells. The epidermis is followed by 5-8 layers of cortical parenchymatous cells, which are more or less compactly arranged and are oval to polyhedral in shape with little or no inter cellular spaces. 3-4 sclerides are present in groups at some places of cortex. The endodermis and pericycle are indistinct. Phloem appears in the form of parenchyma and is interrupted by xylem. Radial vascular bundle is present in the center with equal number of xylem and phloem bundles. Xylem is exarch and the metaxylem vessels meet in the center. The pith is absent. As the growth proceeds (Fig 3.2C and 3.3) the epiblema appears as a single outermost layer of thin walled cells. Outer walls of most of these cells extend outwards and form unicellular root hairs. The epiblema is followed b\ exodermis of 4-5 layers of lignified, thin walled parenchymatous cells arranged 63

4 radially without intercellular spaces representing outer zone of cortex. Outer cortex cells are followed by 8-10 layers of parenchymatous cells, which are i more or less compactly arranged and are oval to polyhedral in shape, without intercellular spacds. In the cortex region, a group of pericyclic fibres are present. The fibres are long, narrow and highly thickened with pointed to blunt ends with narrow; lumen in the center. The phloem consists of sieve tubes, I companion cells and phloem parenchyma. The phloem becomes more wide forming a continuous ring enclosing the xylem. The xylem consists of xylem vessels, tracheids, xylem fibres and xylem parenchyma. Xylem vessels increase in size arid fibres, tracheids and xylem parenchymatous cells become highly thickened and more lignified. The secondary growth starts early in the normal way but phellogen arises in the middle layers of cortex and produces wide cork on its outer side and a very few layers of phelloderm on inner side with the formation of cork. Epidermis ruptures and finally peels off. The mature root is about 8 mm in diameter (Fig. 3.2D, 3.4 arid 3.5) and shows layers of cork cells, which are not uniform in shape, and arrangement of alternate layers of radially elongated and tangentially elongated cells in the outer region as well as in the ; 1 i -i inner region. Besides, the cork cells of inner region are distinctly bigger as i compared to cork cells of middle or outer region. The cork is followed by layers of compressed and tangentially elongated parenchymatous cells showing a small to large intercellular spaces in outer and middle region. Phelloderm cells are intervened by sclereids, which are rounded and highly 64 I

5 thickened with narrow lumen in the center. They are mostly present in groups of The phloem does not show any appreciable development except more phloem parenchymatous cells. Thus, in mature root phloem consists of sieve tubes, companion cells and phloem parenchyma. The xylem is a wide zone and consists of xylem vessels, tracheids, tracheid fibres and xylem parenchyma. All the elements of xylem are highly thickened and lignified. Xylem vessels are mostly distributed singly and at places in groups of two or three. The vessels show mostly bordered pits on their walls. They vary in shape and size being long and cylindrical to short showing transverse to oblique perforated rings. Tracheids are narrow, small mostly bordered and pitted with blunt ends. Fibres vary in size and shape and most of them show highly thickened walls and a narrow lumen except a few with broad lumen. Most of the fibres have pointed ends except the fibres with wide lumen, which show blunt ends. Measurements of different cells in root (in p) Young root: Epidermal cells: 9-12 x 4-20 Cortical cells: 15 x 22 x id root: Exodermis: Cork cells: 2-8x x2-18 Phelloderm cells: 4-7 x 2-8 Phloem parenchyma: 3-7x

6 Xylem vessels: 4-15 Isolated elements: Xylem fibre: x Tracheid: x Xylem parenchyma: x Xylem vessels: x

7 till LIU Fig. 3,2: Microscopy of root of T. purpurea 67

8 Fig. 3.3: Detailed microscopy of matured root of T. purpurea Fig. 3.4: Microscopy of cork portion of root of T. purpurea 68

9 I Fig. 3.5: Microscopy of xylem and phloem of root of T. purpurea 69

10 LEGENDS TO FIGURES (ROOT) Fig. 3.2A T.S of young root (diagrammatic) x 50 Fig. 3.2B Part of TS of Fig 1 showing cellular details Fig. 3.2C TS of slightly matured root (diagrammatic) x 50 Fig. 3.3 Part of TS of fig 3.1C showing cellular details x 450. Fig. 3.2D TS of mature root (diagrammatic) x 50 Fig. 3.4 Part of TS of fig 3. ID showing outermost portion of cork x 1120 Fig. 3.5 Part of TS of fig 3.ID showing inner portion of phloem to outer portion of xylem. Fig-3.2 Isolated elements of root x 100. E-fibres; F-xylem vessels; G-tracheids; H-xylem parenchyma I-Sclerids, CK- cork; Cort-cortex; Epi-epiblema; Phell-phelloderm; Phi-phloem; Scl- sclereids; Tri-Trichomes; Xyl-xylem; XV-xylem vessels; MR-Medullary rays. 70

11 Microscopy of Stem The young stem is about 1 mm in diameter shows more or less rectangular to square outline (Fig.3.6A). The epidermis is single layered covered with thin cuticle. The epidermal cells are elongated to form thick walled covering trichomes with pointed ends, slightly curved with prominent base and thick warty outer surface. These trichomes vary considerably in size. Some are too small appearing as papillae like outgrowth. The upper epidermis is followed by 5-6 layers of collenchymatous cells showing massive thickening on the walls and more or less uniform size and shape with round to oval outline. Beneath the collenchyma parenchyma is represented by 5-9 layers of thin walled round to oval cells with small to large intercellular spaces. The parenchymatous zone is interrupted by patch of sclerenchymatous pericycle, which are highly lignified without intercellular spaces. It is present in the form of arch above the vascular bundle. The phloem is represented by narrow band consisting of sieve tubes, companion cells and phloem parenchyma. Xylem is more than twice in width as compared to phloem and is represented by xylem vessel, tracheids and xylem parenchyma. All the elements show prominent thickening and lignification. Xylem vessels are mostly found singly. The innermost layer is represented by a distinct prominent pith consisting of thin walled round, oval or polyhedral cells with distinct intercellular space. Most of the pith cells show the presence of rhomboidal crystals of calcium oxalate, which are quite big, distinct and characteristic. Some pith cells have small round starch grains.

12 The mature stem (Fig. 3.6B, 3.7 and 3.8) is about 1 cm in diameter arid shows the formation of cork but epidermis and cuticle remains intact except showing tendency to rupture at few place. In the mature stem the outermost layer is single layered epidermis made up of thin rectangular cells and covered externally with prominent cuticle, 3-7 layers of cork cells that are rectangular to square shaped' follow epidermis. They are arranged in radial rows. Cork cells show appreciable suberisation as a consequence of development of the cork. Below the cork wide zone of collenchyma is present followed by parenchyma. The collenchymatous cells become more flattened as a result of pressure of growth and thickening. The parenchymatous cells increase in size and due to pressure of growth they appear tangentially elongated throughout the cortical region showing thin walls and small to mostly large intercellular space. The phloem region consists of sieve tubes, companion cells, phloem parenchyma and phloem fibres. As a result of development phloerii parenchyma are Changed into fibres. The phloem fibres show highly thickened and unlignified walls. They mostly appear groups of two. The phloem fibres are long narrow with pointed to blunt ends and do not show any characteristic shape or feature. The xylem consists of xylem vessels, xylem fibres, tracheids and xylem parenchyma. They all are highly thickened and lignified. The xylem vessels vary in size and shape. They are cylindrical small to moderately long, occasionally narrow and mostly show bordered pits on the walls and transverse to oblique perforated rings. The tracheids are mostly long and all the tracheids show bordered pits on the walls. Xylem fibres vary considerable 72

13 in shape and size with moderate thickening on the walls and possess pointed to blunt ends. Xylerri parenchymatous cells are mostly rectangular in shape but some show irregular outline. All the xylem parenchymatous cells show single pits on the walls. Below xylem a small group of phloem called inter-xylary or included phloem is seen which arises centripetally in the xylem due the irregular activity of cambium. These groups are entrapped in the xylem when the stem begins to mature. The central region of the stem shows prominent pith, which are round or oval to isodiametric cells with distinct intercellular spaces. Acicular crystals of calcium oxalate are seen unlike the rhomboid ones in the young stem. Measurements of different cells in Stem (in u) i Epidermal cells: 10-25x9-31 i Cortical collenchyma: 7-32 x 7-26 Cortical parenchyma: x Phloem: 3-7 Xylem vessels: 7-58 Pith cells: ; x Isolated elements: 1 Xylem fibre: i x17-22 Phloem fibre: x Xylem vessels: x 7-57 Tracheids: : x Calcium oxalate crystals:

14 Fig. 3.6: Microscopy of stem of T. pupurea I 74

15 Fig. 3.7: Microscopy of matured stem of T. purpurea 75

16 LEGENDS TO FIGURES (STEM) Fig. 3.6A T.S of young stem (diagrammatic) x 50 Fig. 3.6B TS of advance stage of young stem (diagrammatic) x 50 Fig. 3.7 TS of matured stem x 50 Fig. 3.8 Innermost portion of xylem, interxylary phloem and pith. Fig. 3.6 Isolated elements of stem x 100. C-Trichomes; D- phloem fibres; E- xylem fibres; F-xylem-vessels; G- tracheids; H-Starch grains; I-Calcium oxalate crystals; J- xylem parenchyma. CK-cork, Cort-cortex, Epi-epidermis, Phi-phloem, Scl-sclereids, Xyl-xylem, Tri-Trichome; Hyp-Hyperdermis; Ixp-Inter xylary phloem. Pi-Pith.

17 Microscopy of Leaf The midrib and mesophyll region shows isobilateral appearance (Fig. 3.9). The microscopy of the mid rib portion showed upper epidermis which is single layered covered distinctly by a thick cuticle. The epidermal cells are mostly rectangular and radically elongated. Trichomes are absent on the upper epidermis. The upper epidermis is followed by 2-5 layers of collenchymatous cells showing thickening on their walls. They are round to oval in outline. Beneath collenchyma, parenchymatous cells are represented by 5-8 layers of thin walled round to oval with small to large inter cellular spaces. The lower epidermis consists of single layer of thin walled cells having rectangular or square shape covered externally by thickened cuticle. Many cells are converted to unicellular covering trichomes, having blunt ends, which are covered by thick cuticle. Above the lower epidermis the collenchyma is present as a narrow zone and consists of 2-5 layers of cells with highly thickened walls. The central region of the mid rib is represented by crescent shaped vascular bundle. The vascular bundle is represented by wide zone of xylem elements comprising of radiating rows of thick walled xylem cells alternating with thin walled xylem parenchymatous cells. The xylem is surrounded by strands of phloem. The xylem consists mainly of vessels and tracheids, which are thick walled and lignified and thin walled parenchymatous cells. However the phloem consists of sieve tube, companion cells and phloem parenchyma. In the mesophyll region the upper epidermis and lower epidermis is similar to mid rib region. The upper epidermis is followed by a single layer of! 77

18 palisade cells, which are mostly tubular and distinctly radially elongated. The palisade cells are followed by 3-4 layers of spongy paranchymatous cells showing large intercellular spaces. The spongy parenchyma varies in size, shape and outline. However, at places, spongy parenchyma is interrupted by xylem vessels arranged in groups showing spiral thickening on their walls. In the lower epidermis in the surface view showed paracytic stomata are seen. Measurements of different cells in Leaf (in p) Upper Epidermal cells: x 7-21 Lower Epidermal cells: x Collenchymatous cells: Ventral side: x Dorsal side: 7-26 x 7-24 Paranchymatous cells: x Trichomes: x 7-23 Upper palisade: 33-40x6-10 Lower palisade: x 6-9 Spongy parenchyma: x LEGENDS TO FIGURES (LEAF) Fig. 3.9A T.S of leaf (diagrammatic) x 50 Fig. 3.9B Detailed TS of leaf x 100 UC-Upper collenchyma; LC-Lower collenchyma; UE-Upper epidermis; LE-Lower epidermis; UP-Upper palisade; LP-Lower palisade; SP-Spongy parenchyma; Xyl- xylem; Phl-phloem; Tri-Trichome;

19 UE UP SP LP Fig. 3.9: Microscopy of leaf off. purpurea 79

20 Syzygium cuminii (Linn) Skeels Syzygium cuminii (Linn) fruits (Fig. 3.10) are ellipsoid or oblong, up to cm long black with pinkish juicy pulp and the seeds were single, shaped like the fruit, sometimes 2-4 seeds compressed together into a mass resembling a single seed, the whole enclosed in cream colored coriaceous covering, smooth, oval or round and taste astringent. Fig. 3.10: Fruits of Syzygium cuminii 80

21 Microscopy of seeds The microscopy of seeds (Fig. 3.11) showed cotyledons consisting of single layered epidermis, endosperm consists of isodiametric, thin walled, paranchymatous cells fully packed with simple starch grains which are oval or round measuring 7-28 p in diameter and few schizogenous cavities are also found Fig. 3.11: Microscopy of seeds of Syzygium cuminii 81

22 PROXIMATE ANALYSIS Proximate analysis aids to set up certain standard for dried crude drugs in order to avoid batch-to-batch variation and also to judge their quality. These studies also give an idea regarding the nature of phytoconstituents present. Proximate analysis of these crude drug powders was carried out using reported methods by subjecting them to various determinations. The different values obtained from the proximate analysis of the selected drugs are recorded in Table 3.1, 3.2, 3.3 and Foreign matter: An accurate quantity of the sample was weighed and spread it a thin layer and foreign matter was sorted into groups by visual inspection. The portions of the sorted foreign matter was weighed and the content of foreign matter in each drug in grams per loog of air-dried sample was calculated. 2. Moisture content / Loss on drying: lg of the crude drug was accurately weighed in an oven dried bottle and this bottle was placed after removing the lid in a dessicator over phosphorous pentoxide at room temperature till constant weight was attained. The moisture content was determined in terms of % w/w. 3. Total Ash value; 2.0 g of the air dried sample of crude drug was weighed accurately in. a tarred silica crucible and incinerated below 450 C until free from carbon, cooled and reweighed. The percentage of ash with reference to the air-dried drug was calculated. 32

23 4. Acid-insoluble Ash value: The ash obtained in total ash was boiled with 25 ml 2N Hydrochloric acid for five minutes, the insoluble matter collected in a gouch crucible, which was then washed with hot water. The insoluble matter was ignited and weighed. The percentage of acid-soluble ash with reference to the air-dried drug was calculated. 5. Water-soluble Ash value; The, ash obtained in total ash was boiled for 5 minutes with 25ml of water, collected insoluble matter in a Gooch crucible and washed with hot water and ignited for 15 minutes below 450 C. weight of the insoluble matter was subtracted the from the weight of the ash. The percentage of water-soluble ash with reference to the air-dried drug was calculated. 6. Sulphated Ash value: A silica crucible was heated to redness for 10 minutes and allowed to cool in a dessicator and was weighed. 1 g of the crude drug was transferred into this crucible and was gently ignited, until the substance was thoroughly charred and was then cooled. The residue was moistened with 1ml of Sulphuric acid, heated gently until white fumes are no longer evolved and ignited at 800 C until all black particles have disappeared. The ignition was conducted in a place protected from air currents. The crucible was allowed to cool and added a few drops of sulphuric acid and heated. The contents were ignited as before and allowed to cool and weighed. 83

24 7. Acid Soluble extractive value: 5.0 g of air-dried coarsely powdered crude drug was macerated with 100 ml of absolute alcohol in a closed flask for 24 hours shaking frequently during 6 hours and allowed to stand for 18 hours. Filtered rapidly taking precautions against loss of alcohol, evaporated 25ml of filterate to dryness in a tarred flat shallow dish dried at 105 C and weighed. The percentage of alcohol-soluble extractive with reference to the air-dried drug was calculated. 8. Water Soluble extractive value 5.0 g of air-dried coarsely powdered crude drug was macerated with 100 ml of Chloroform water in a closed flask for 24 hours shaking frequently during 6 hours and allowed to stand for 18 hours. Filtered rapidly, evaporated 25ml of filterate to dryness in a tarred flat shallow dish dried at 105 C and weighed. The percentage of alcohol-soluble extractive with reference to the air-dried drug was calculated. PHYTOCHEMICAL STUDIES: 200g each of powdered drug was successively extracted with the solvents of increasing polarity i.e. petroleum ether (40-60 C) benzene, chloroform, ethyl acetate and alcohol (95%). Finally the drug was subjected to maceration with distilled water for 48 hours. The completion of the extraction was confirmed by evaporating a few drops of the extract from the thimble on a watch glass to observe that no residue remained after evaporation of the solvent. The liquid extracts obtained with different solvents were collected. 84.

25 After each extraction, the exhausted drug was kept in open to evaporate the solvent. After drying it was packed again in the Soxhlet s apparatus with next solvent. The extracts were concentrated by distillation until thick viscous residue remained in the distillation flask. The extracts were evaporated to dryness in oven at 50 C and weighed. The percentage (% w/w) of the dry extract was calculated on the basis of the material (Table 3.3 and 3.4) and then subjected to qualitative chemical examination for various phytoconstituents as follows Testing of extracts: Test for alkaloids: About 1 ml of each concentrated extracts were evaporated to dryness at low temperature and the residue was taken in 5% HC1 (2ml) solution. The filtrates were tested with different alkaloidal reagent such as, Dragendroff s, Hager s and Wagner s reagent. Test for sterol and terpenes: Libermann Burchard s Reaction: 10 ml of each concentrated extracts were evaporated to dryness under vacuum and the residue was dissolved in dry chloroform (0.5 ml) in the test tube and acetic anhydride (0.5 ml) Result of qualitative chemical tests are reported in Table 3.5 and 3.6. The TLC studies were also performed using various solvent systems [Table 3.7] used on the plates coated with the silica gel G [S.D. Fine Chemicals] and the results are reported in Table 3.8 and

26 Table 3.1: The percentage yield of dry extract of aerial parts of Tephrosia pupurea. Solvent Color of the extract Consistency Weight (g) Dry extract (% w/w) Pet. Ether Green Semi solid Benzene Green Semi solid Chloroform Dark green Solid Ethyl acetate Brown Solid Ethyl alcohol Brown Solid Water Black Solid Table 3.2 : The percentage yield of dry extract of Syzygium cuminii seeds Solvent Color of the extract Consistency Weight (g) Dry extract (% w/w) Pet. Ether Brown Semi solid Benzene Red Solid Chloroform Red > Solid Ethyl acetate Pale Yellow Solid Ethyl alcohol Dark yellow Solid Water Brown Solid

27 Table 3.3: Proximate analysis of aerial parts of Tephrosia purpurea S. No Parameter Determination [%w/w] 1 Foreign matter Moisture content Total Ash Acid insoluble Ash Water soluble Ash Sulphated ash Alcohol soluble extractive Water soluble extractive 5.43 Table 3,4: Proximate analysis of Syzygium cuminii seeds S. No Parameter Determination [%w/w] 1 Foreign matter Moisture content Total Ash Acid insoluble Ash Water soluble Ash Sulphated ash Alcohol soluble extractive Water soluble extractive

28 Table 3.5: Qualitative chemical test of different extracts of aerial parts of Tephrosia purpurea. Constituents P B C E A W 1. Alkaloids: a. Dragendorff s test b. Hager s test c. Wagner s test 2. Carbohydrates and Glycoside a. Molisch s test b. Fehling s test c. Benedict s test d. Legal s test 3. Steroids & terpenes Fixed oil & fat a. Spot test b. Saponification test * m 5. Saponins a. Foam test - b. Haemolytic test Phenolic compound & Tannins - - a. Ferric Chloride test : b. Potassium chromate test c. Lead acetate test d. Gelatin test e. Aq. Bromine test Protein a. Millon s test b. Xanthoproteic test c. Ninhydrin test Test for Flavonoid * 88

29 Table 3.6: Qualitative chemical test of different extracts of Syzygium cuminii seeds Constituents P B C E A W 1. Alkaloids: d. Dragendorffs test e. Hager s test f. Wagner s test Carbohydrates & Glycoside e. Molisch s test f. Fehling s test g. Benedict s test J_ h. Legal s test Steroids & terpenes + 4~ Fixed oil & fat c. Spot test d. Saponification test Saponins c. Foam test d. Haemolytic test " - 6. Phenolic compound & Tannins f. Ferric Chloride test g. Potassium chromate - - -, test h. Lead acetate test i. Gelatin test j. Aq. Bromine test Protein d. Millon s test e. Xanthoproteic test f. Ninhydrin test Test for Flavonoid

30 Table 3.7: Solvent systems used to perform the Thin Layer Chromatography studies Solvent system Solvents used Ratio A Benzene: Chloroform 1 : 1 B Ethyl acetate : Alcohol 1 : 1 C n-butanol : Ammonia : Water 9.1 : 0.1 : 0.8 D Ethyl acetate : Chloroform 1:1 E Alcohol: Chloroform 1 : 1 F Chloroform : Methanol 1.2: 0.8 G Chloroform : Methanol: Acetic acid 6.0: 1.0: 0.1 H. Benzene : Ethyl acetate 1:1 I Butanol: Acetone : Water 6.1 : 1.5 : 1.5 J Toluene : Ethyl acetate 9 : 1 90

31 91 Water Ext. 2 (0.4, 0.7) 2(0.5,0.75) (0.2, 0.6, 0.9) r (0.7, 0.9) 3 (0.3, 0.7, 0.9) 2 (0.63, 0.95) 3 (0.3, 0.66, 0.8) 3 (0.45, 0.7, 0.9) Alcohol Ext. 2(0.33,0.65) 2 (0.4, 0.7) (0.15, 0.5, 0.9) (0.6, 0.92) 3 (0.2, 0.54, 0.9) 2 (0.55, 0.90) 3 (0.3, 0.5, 0.66) 3 (0.4, 0.7, 0.9) Table 3.8: Thin layer chromatographic pattern of different extracts of Tephrosia purpurea. Number of spots (Rf value) Eth. acetate Ext. Chloroform Ext.! Benzene Ext. Pet. Ether Ext Solvent system i (0.30, 0.52) i I 1 1 l 2 (0.40, 0.75) 4 (0.25, 0.40, 0.58, 0.80) 1 ' ' 1 l (0.80) ooc o I I 2 (0.7, 0.9) 2 (0.55, 0.8) l 1 I I 2 (0.45, 0.65) 2 (0.3, 0.55) i 2 (0.5, 0.8) 1 < CQ U Q w tt-i O S3 HH h s 1

32 92 Water Ext. Tt O r i 1 2 (0.7, 0.9) 2 ( 0.4, 0.7) 2 (0.3, 0.55) 2 (0.5, 0.85) 1 3( 0.7, 0.9) 2 (0.36, 0.8) Alcohol Ext. 2 (0.5, 0.75) > ( 1 2 (0.5, 0.9) 3(03,0.4,0.8) 2 (0.4, 0.9) 2 (0.3, 0.5) 2 (0.6, 0.8) 2 (0.5, 0.9) 3 (0.2, 0.8, 0.9) Table 3.9: Thin layer chromatographic pattern of different extracts of Syzygium cuminii Number of spots (Rf value) Eth. acetate Ext. Chloroform Ext. Benzene Ext. Pet. Ether Ext Solvent system 1 (0.7) 2 (0.6, 0.8) 2 (0.4, 0.6) 2(0.60,0.80) ' i 1!! I 1 ' 1 ' 1 1 ' 2 (0.55, 0.9) 3 (0.2, 0.58, 0.80) 2 (0.6,0.8) 2 (0.4, 0.75) 2 (0.5, 0.8) 3 (0.4, 0.75, 0.8) < CQ U Q W O X > < 1 2 (0.55, 0.8) l