|Year : 2018 | Volume
| Issue : 2 | Page : 70-76
Bioactivity and chemical constituents of Gleditsia triacanthos L. leaves
Aisha H Abou Zeid1, Seham S El-Hawary2, Wedian E Ashour1, Reda S Mohammed1, Amany A Sleem3
1 Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo, Egypt
2 Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
3 Department of Pharmacology, National Research Centre, Giza, Egypt
|Date of Submission||19-Dec-2017|
|Date of Acceptance||08-Mar-2018|
|Date of Web Publication||6-Sep-2018|
Reda S Mohammed
Pharmacognosy Department, National Research Centre, Giza 12622
Source of Support: None, Conflict of Interest: None
Background and objective Gleditsia triacanthos L. is a deciduous tree belonging to the family Fabaceae. It is used in folk medicine as it possesses important biological activities: antimutagenic, anticancer, cytotoxic, and rheumatoid arthritis-treating properties. The study aimed to evaluate the anti-inflammatory, analgesic, and antipyretic activities of the nonpolar extract of the plant in addition to investigation of its chemical composition.
Materials and methods The dried powdered leaves of G. triacanthos were successively extracted with solvents of increasing polarities: petroleum ether (PE), chloroform, ethyl acetate, and aqueous ethanol; moreover, the total ethanol extract was also prepared. The PE extract was fractionated into unsaponifiable matter (USM) and fatty acids, which were methylated to give FAME fractions, which are subjected to GC/MS analysis.
Results GC/MS analysis of USM and FAME revealed that 24 compounds represented 76.86% of the total fraction of USM, with squalene (22.68%) as the major compound followed by nonacosane (21.03%) and isophytol (14.70%). Twenty-one compounds of FAME fraction representing 85.87% were identified, with methyl 9,12,15-octadecatrienoate (31.37%) as the major compound followed by methyl hexadecanoate (19.35%) and methyl 9,12-octadecadienoate (13.52%). The unsaturated fatty acid represented 45.89% of the total fraction. GLC of the sterol fraction isolated from the PE revealed the identification of campesterol (51.23%), stigmasterol (42.34%), β sitosterol (4.88%), and cholesterol (1.55%). The PE extract exhibited the highest analgesic and antipyretic activities (81.73 and 91.10% potency, respectively) in comparison with tramadol and paracetamol (100% potency), correspondingly, whereas total ethanol extract exhibited the highest anti-inflammatory activity followed by ethyl acetate and PE extract (74.60, 66.66 and 62.69% potency, respectively) in comparison with indomethacin (100% potency).
Conclusion The results of our study suggest that the PE extract of G. triacanthos leaves extracts can be used as an alternative therapy for their analgesic, anti-inflammatory, and antipyretic activities.
Keywords: analgesic antipyretic and anti-inflammatory activities, fatty acids, Gleditsia triacanthos, unsaponifiable matter
|How to cite this article:|
Abou Zeid AH, El-Hawary SS, Ashour WE, Mohammed RS, Sleem AA. Bioactivity and chemical constituents of Gleditsia triacanthos L. leaves. Egypt Pharmaceut J 2018;17:70-6
|How to cite this URL:|
Abou Zeid AH, El-Hawary SS, Ashour WE, Mohammed RS, Sleem AA. Bioactivity and chemical constituents of Gleditsia triacanthos L. leaves. Egypt Pharmaceut J [serial online] 2018 [cited 2018 Nov 21];17:70-6. Available from: http://www.epj.eg.net/text.asp?2018/17/2/70/240671
| Introduction|| |
Gleditsia triacanthos L. (Honey locusts) is a deciduous tree belonging to the family Fabaceae. It flowers in July, and the seeds ripe from October to November. The plant comprises 12 species , and it can reach a height of 20–30 m; their life spans are ∼120 years. The Honey locust is not a honey plant; the name was derived from the sweet taste of the legume pulp, which was used for food by native American people, and it can be fermented to make beer. The pulp on the insides of the pods is edible, but the black locust is toxic. Gleditsia spp. have been used in folk medicine, and the anomalous fruits produced by old or injured plants of Gleditsia sinensis L have long been known in traditional Chinese medicine as saponin-rich herbal medicine and as pesticide . G. triacanthos has been used in folk medicine: pods have been used as a folk remedy for dyspepsia, measles, indigestion, and as antiseptic; bark for dyspepsia and in the treatment of whooping cough, measles, and smallpox; seeds as a natural source of phenolic compounds and antioxidants; and leaves used as a potential source of anticancer compounds . G. triacanthos was reported to contain triterpene glycoside triacanthoside C in addition to oleanolic and echinocystic acids isolated from the pericarps  gleditschosides A, B, C, D, and E were detected in pericarp and leaves of G. triacanthos . Different Gleditsia fruits species were found to contain saponin triacanthosides A1, G, and C together with gleditschosides A, B, C, D, and E, which were isolated from G. triacanthos ,,,. Triacanthine alkaloid was isolated from the leaves of G. triacanthos ,. Vitexin, luteolin, isovitexin, and quercetin are reported to be isolated from G. triacanthos ,. G. triacanthos leaves were studied for their flavonoid content and cytotoxic and antioxidant activities . In this view, it is imperative to carry out more pharmacognostical and pharmacological investigations on G. triacanthos leaves to find out the unexploited potential of this medicinal plant under study. This plant is chosen for study because it is in abundance, easily available, safe, and widely used in traditional medicine.
| Materials and methods|| |
Silica gel 60 for column chromatography (E. Merck, Darmstadt, Germany), aluminium sheets G60 F254 (Silica gel, layer thickness 0.2 mm; Fluka Chemie AG, Switzerland); solvent system: benzene–acetone (9 : 1 v/v); chloroform (Ch)–methanol (9.5 : 0.5 v/v); benzene–ethyl acetate (EA) (3 : 1 v/v), reagent for detection sterols and terpenes: 1% vanillin–sulfuric acid reagent. Apparatus for GC/MS analysis of unsaponifiable matter (USM) FAME was gas chromatograph coupled with a mass spectrometer (Agilent 6890 gas chromatograph coupled with an Agilent mass spectrometric detector, 70 eV). Agilent Technologies 6890 N (Network GC System, USA) was used for GLC analysis of sterol fraction. Indomethacin (Kahira Pharmaceuticals Industries Company, Cairo, Egypt) was used as a standard anti-inflammatory agent; carrageenan (Sigma Co.) was used for induction of acute inflammation in rats; paracetamol was used as a standard antipyretic drug and tramadol as a standard analgesic drug (Misr Co. for Pharmaceuticals Industries, Cairo, Egypt). Solvents used were petroleum ether (PE) (BP 60–80°C), benzene, diethyl ether, EA, Ch, dichloromethane, 70% and 95% ethanol, methanol, and distilled water. Solvents were distilled and dehydrated before use.
Conditions of GC/MS analysis
Capillary column of Thermo Scientific TR-5MS (5% phenyl polysilphenylene siloxane), 30 m length, 0.25 mm, internal diameter, and 0.25 μm thickness, was used. Carrier gas was helium at 13 psi, injector temperature was 200°C, detector temperature was 280°C, and temperature programming was 60°C for 5 min, 60–280°C at a rate of 5°C/min, and 280°C isothermal for 10 min for USM and 5 min for FAME. The detector was mass spectrometer.
Conditions of GLC analysis
The conditions for GLC analysis were as follows: capillary column HP-5 (5% phenyl methyl siloxan), 30 m length, 0.32 mm, internal diameter, and 0.25 μm thickness, initial temperature:80°C, initial time: 1.00 min, rate: 8°C/min, final temperature: 300°C, inlet temperature: 250°C, detector: 300°C flame ionization detector (FID), flow: 2 ml/min, carrier gas: N2 30 ml/min, H2 30 ml/min, and air 300 ml/min.
Fresh leaves of G. triacanthos L. were obtained from the Zoo in May 2007. The plant was identified by Terase Labib, the plant taxonomist of Orman Garden, Giza, Egypt, and confirmed by the taxonomist Dr M. El-Gebaly, NRC. A voucher specimen (M 96) was deposited by Dr Mona Marzouk and kept in the herbarium of Pharmacognosy Department, NRC.
In this study, adult albino rats of Sprague Dawley strain weighing 130–150 g and albino mice weighing 25–30 g were used. Animals were obtained from the Animal House Colony of the National Research Centre, Dokki, Egypt. They were kept under the same hygienic conditions with well-balanced water and diet. Normal diet consisted of vitamin mixture 1%, mineral mixture 4%, corn oil 10%, sucrose 20%, cellulose 0.2%, and casein (95% pure) 10.5%, and starch 54.3%. Medical research ethical committee in the National Research Center has approved the work (09/130) 2009.
Investigation of nonpolar extract
Preparation of plant extracts
The different extracts of the dried powdered leaves were previously prepared as reported .
Preparation of the USM and FAME
Five grams of the PE extract of the leaves was subjected to saponification, and the USM and the FAME were prepared adopting the reported methods  to give 2.5 g of the USM and 1.5 g of the fatty acids (FAs).
GC/MS analysis of USM matter and FAME
The USM matter and FAME were subjected to GC/MS analysis adopting the aforementioned conditions. The identification of the compounds was accomplished by comparing their retention times and mass spectral data with those of the library and published data ,. Quantitative determinations were carried out based on peak area measurements. The results are listed in [Table 1] and [Table 2].
|Table 1 Results of GC/MS analysis of the USM of PE extract of G. triacanthos L. leaves|
Click here to view
|Table 2 Results of GC/MS analysis of FAME of PE extract of G. triacanthos L. leaves|
Click here to view
Isolation of the sterol fraction by column chromatography
Five grams of the PE extract was subjected to fractionation on silica gel column (G 60 F 254; Merck) using PE (60–80)/Ch as the mobile phase. Ten fractions were collected and monitored by TLC in comparison with authentic β-sitosterol using benzene–acetone (9 : 1 v/v) solvent system spraying with 10% H2SO4 and heating in oven at 110°C. The fractions proved to contain β-sitosterols (Rf 0.5) were mixed and subjected to preparative TLC using the same solvent system. The sterol band was scratched and eluted by Ch and then analyzed by GLC analysis ([Table 3]).
|Table 3 Results of GLC analysis of sterol fraction of G. triacanthos L. leaves|
Click here to view
Acute toxicity test (LD50)
The total ethanol (TE) extract of G. triacanthos leaves was subjected to LD50 determination according to the reported method . Male albino mice (25–30 g) were divided into groups of six animals each. Preliminary experiments were done to determine the minimum dose that kills all animals (LD100) and the maximum dose that fails to kill any animal. Several doses at equal logarithmic intervals were chosen in between these two doses, and each dose was injected subcutaneously into a group of six animals. The mice were then observed for 24 h, and symptoms of toxicity and mortality rates in each group were recorded and LD50 was calculated.
Analgesic activity test
The analgesic activity was determined by acetic acid-induced writhing method using Swiss albino mice (20–25 g). Animals were acclimatized to the laboratory conditions for at least 1 h before testing and were used once during the experiment. Forty-eight albino mice were divided into eight groups, with six animals each. First group albino mice that received 1 ml saline serving as a control, second and third group mice received 50 and 100 mg/kg, body weight, of TE extract. Fourth, fifth, sixth, and seventh groups mice received 100 mg/kg, body weight, of PE, Ch, EA, and aqueous ethanol (AE) extracts, respectively. Eighth group mice received 4 mg/kg, body weight, of the standard analgesic (tramadol), and 30 min later 0.6% acetic acid was injected intraperitoneally (0.2 ml/mice). Each mouse was then placed in an individual clear plastic observed chamber, and the total number of writhes/30 min was counted for each mouse . The numbers of writhing and stretching were recorded and compared with the control drug. The results were illustrated in [Table 4].
|Table 4 Analgesic activity of TE and successive extracts of G. triacanthos L. leaves|
Click here to view
The percent was calculated using the following ratio:
Antipyretic activity test 
Forty-eight male albino rats, with average body weight of 100 g, were divided into eight groups, with six animals each. The normal rectal temperature was recorded before starting the experiment. Pyrexia was induced by intramuscular injection of 1 ml/100 g, body weight of 44% yeast suspension. The site of injection was massaged to spread the suspension beneath the skin. After 18 h, the rectal temperature was recorded for all groups to serve as the baseline of elevated body temperature, to which the antipyretic effect will be compared. A single oral administration of a dose of each tested extract, paracetamol or saline, was given. One and two hours later, other records of rectal temperature were measured and the results are compiled in [Table 5].
|Table 5 Antipyretic activity of TE and successive extracts of G. triacanthos L. leaves|
Click here to view
Anti-inflammatory activity test
The anti-inflammatory test was evaluated adopting the carrageenan-induced rat hind-paw edema test . This model uses carrageenan as an irritant to induce paw edema. NSAIDs such as indomethacin, reduce paw swelling in a dose-dependent manner to a maximum of 60%. Test materials are assessed for acute anti-inflammatory activity by examining their ability to reduce or prevent the development of carrageenan-induced paw swelling. Forty-eight adult male albino rats, divided into eight groups, with six animals each, were orally treated with one of the following: 50 and 100 mg/kg body weight of the TE extract, 100 mg/kg body weight of each successive extract, 20 mg/kg body weight of indomethacin, and saline (control), as shown in [Table 6]. One hour after oral administration, all animals were given a subplantar injection of 0.1 ml of 1% carrageenan solution in saline in the right hind paw and 0.1 ml saline in the left hind paw. Four hours after oral administration, the rats were killed. Both hind paws were, separately, excised and weighed. Edema percentage and edema inhibition percentage were calculated according to the following equations. Results are illustrated in [Table 5].
|Table 6 Anti-inflammatory activity of TE and successive extracts of G. triacanthos L. leaves|
Click here to view
Mc: the mean edema in the control group; Mt: the mean edema in the drug-treated group.
The results of the aforementioned three experiments were statistically analyzed using the Student’s t-test . Results with P value of less than 0.01 were considered statistically significant.
| Results|| |
GC/MS analysis of the USM of PE extract of G. triacanthos leaves ([Table 1]) revealed identification of 24 compounds constituting 76.86% of the total composition. The oxygenated compounds constituted 23.28% whereas the nonoxygenated compounds constituted 53.58%. Squalene (22.68%), n-nonacosane (21.03), and isophytol (14.70%) were the major compounds. Analysis of the FA methyl esters by GC/MS ([Table 2]) revealed the identification of 21 compounds representing 85.87% of the total FA, the unsaturated FAs constituted 45.89%, whereas the saturated constituted 39.98%. 9, 12, 15-Octadecatrienoic acid was the major polyunsaturated compound (31.37%), followed by hexadecanoic acid (19.35%), and 9,12-octadecadienoic acid (13.52%). Campesterol (51.23%), stigmasterol (42.34%), β sitosterol (4.88%), and cholesterol (1.55%) were identified by GLC analysis of the sterol fraction of the PE extract ([Table 3]).
LD50 of the TE extract of leaves of the plant was found to be 6.6 g/kg, body weight (the plant is safe); this explains the use of different parts (seeds, young leaves and fruits) of the plant as food in some countries ,,. The highest analgesic activity was exhibited by 100 mg of PE, AE, TE, Ch, and EA (81.73, 77.40, 68.43, 64.45 and 59.47% potency, respectively) in comparison with tramadol (100% potency).
The PE (100 mg) exhibited significant antipyretic activity after 2 h (91.10% potency) followed by 100 mg of Ch, TE, and EA (69.69, 56.85 and 56.85% potency, respectively) in comparison with 20 mg of paracetamol (100% potency). The AE exhibited very low activity. All extracts exhibited anti-inflammatory activity as indicated by inhibition of the rat paw edema weight induced by carrageenan. The significant anti-inflammatory activity was found to be dose dependent and the highest activity was found to be exhibited by 100 mg of the TE, EA, PE, AE, and Ch (74.6, 66.66, 62.69, 56.34 and 51.84% potency, respectively) in comparison with indomethacin (100% potency).
| Discussion|| |
Pain is one of the basic symptoms of all human ailments and is a sensorial modality and primary protection response; analgesics relieve pain without affecting its cause . Acetic acid writhing test along with electrical stimulation of the tail of the mice is a sensitive way to measure the analgesic activity . The normal body temperature of an individual is controlled by hypothalamus gland which regulates rising or decreasing body temperature through adjusting the balance between heat production and heat loss. So any disturbance of hypothalamic thermostat results in a complaint of fever . Pyrexia or fever is caused as a result of infection, tissue damage, inflammation, graft rejection, malignancy, or other diseased states. It is the body’s natural defense to create an environment where infectious agent or damaged tissue cannot survive. The infected or damaged tissue initiates the formation of proinflammatory mediators (cytokines like interleukin 1β, α, β, and tumor necrosis factor-α). Yeasts induce pathogenic pyrexia, which leads to the production of PGE2 that affects the thermoregulatory center, leading to elevation of body temperature . So the principal action of antipyretics is the ability to inhibit the enzyme cyclooxygenase (COX) and interrupt the synthesis of inflammatory prostaglandins . Moreover, the synthetic antipyretic agents irreversibly inhibit COX-2 with high selectivity but are toxic to the hepatic cells, glomeruli, cortex of brain, and heart muscles, whereas natural COX-2 inhibitors have lower selectivity with fewer adverse effects . In our study, the plant extracts may reduce pyrexia by reducing brain concentration of prostaglandin E2, especially in the hypothalamus. Inflammation is a complex biological response of vascular tissues to harmful stimuli. The primary signs of acute inflammation were pain, heat, redness, and swelling, and may lead to loss of function . Inflammation is the first physiological defense system which involves the activation of many enzymes such as COX-1 and COX-2 and lipooxygenase, which are responsible for the release of inflammatory mediators (prostaglandins and leukotrienes, which in turn lead to fluid exudation, cell migration, and tissue breakdown) ,. Phytochemical screening of PE extract revealed presence of sterols and/or triterpenes. The unsaponifiable fraction contains appropriate percentage of α-tocopherol, β amyrin, and isophytol. α-Tocopherol has been shown to have anti-inflammatory effects both in vitro and in vivo, and the use of high doses of α-tocopherol has been shown to decrease release of proinflammatory cytokines. β-Amyrin is a triterpenoid and was found to be the precursor of boswellic acid and ursolic acid which have strong 5-lipooxygenase inhibitor and antielastase activity. Phytol and isophytol are volatile terpenoids reported for the treatment of rheumatoid arthritis and other chronic inflammatory diseases . 9, 12, 15-Octadecatrienoate (linolenic acid) is the major compound in the saponifiable fraction. It is an ω-3 (18 : 3, n-3) FA which metabolized in the body to octadecatetraenoic acid (18 : 4, n-3), stearadonic acid (20 : 4, n-3), and eicosapentaenoic acid (20 : 5, n-3). The anti-inflammatory properties of ω-3 FAs, especially eicosapentaenoic acid, are due to competition with arachidonic acid (AA) as a substrate for COX and 5-lipoxygenase . At present, the natural analgesics and anti-inflammatory agents of plant origin are being appreciated owing to possible toxicity of synthetic drugs. The results of this study suggest that G. triacanthos leaves can be used as a supplementary or alternative therapy for their analagesic, anti-inflammatory, and antipyretic activities.
The authors extend special appreciation to the National Research Centre, Dokki, Giza, Egypt for offering the facilities for this study. This work was extracted from the MSc Thesis of Wedian El-Sayed Mohamed Ashour, Pharmacognosy Department, National Research Centre.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Bailey LH, Bailey EZ. Hortus Third: A Conise Dictionary of Plants Cultivated in the United States and Canada. New York, NY: Macmillan Publishing Co. Inc; 1976. p. 513.
Zhong J, Da Y, Dian C. New Medical College Shanghai People's Public Health Publishing House: Shanghai, 1977. pp.1144, 1145, 1147, 2198.
Institutum Botanicum Kunmingesnse Academiae Sinicae Edita. Index Florae Yunnanensis, Tomus I. Kunming: People’s Publishing House; 1984. p. 556.
Badalbaeva TA, Kondratenko ES, Mzhel’skaya LG, Abubakirov NK. Triterpene glycosides of Gleditschia triacanthos. II. Structure of triacanthosides C
. Khim Prir Soedin 1972; 6:741–744.
Badalbaeva TA, Kondratenko ES, Mzhel’skaya LG, Abubakirov NK. Triterpene glycosides of Gleditschia triacanthos
III. The structure of triacanthosides G. Khim Prir Soedin 1972; 6:744–747.
Badalbaeva TA, Kondratenko ES, Abubakirov NK. Triterpene glycosides of Gleditschia triacanthos.
V. Structure of triacanthosides C and the minor glycosides. Khim Prir Soedin 1973; 5:635–640.
Badalbaeva TA, Kondratenko ES, Abubakirov NK. Triterpene glycosides of Gleditschia triacanthos
.IV. Structure of triacanthosides A1 and G. Khim Prir Soedin 1973; 9:314–316.
Panova DI, Georgieva ES, Stanoeva E. Components of Gleditschia triacanthos
. 2. Isolation of triacanthine and detection of hydrocarbons and alcohols. Pharmazie 1971; 26:493–494.
Masterová I, Grznár K, Tomko J. Monitoring the triacanthine content during development in the leaves of Gleditsia triacanthos
L. Cesk Farm 1977; 26:305–307.
Panova DI, Georgieva ES. Study of the flavonoid composition of Gleditschia triacanthos
leaves. Dokl Bolg Akad Nauk 1972; 25:71–74.
Leibovici B, Petrovanu M, Lazar M, Segal B. Determination of flavones components from Gleditschia triacanthos
. Rev Chim 1986; 37:81–82.
Mohammed RS, Abou Zeid AH, El Hawary SS, Sleem AA, Ashour WE. Flavonoid constituents, cytotoxic and antioxidant activities of Gleditsia triacanthos
L. leaves’. Saudi J Biol Sci 2014; 21:547–553.
Abou Zeid AH, Hifnawy MS, Mohammed RS, Sleem AA. Lipoidal contents, analgesic and antipyretic activities of the aerial parts of Dichrostachys cinerea
L. J Herbs Spices Med Plants 2015; 21:118–128.
Adams RP. Identification of essential oil compounds by gas chromatography/mass spectroscopy. Carol Stream, Illinois, USA: Allured Publishing Corporation; 2009.
Eight Peaks Index of Mass Spectra 1980 2nd ed. Published by Mass Spectrometry Data Centre, AWRE Aldermaston, Reading, UK.
Abou Zeid AH, El-Kashoury EA, Sleem AA, Waly DA. Lipoidal content and anti-inflammatory activity of Adenanthera pavonina
L. leaves. J Appl Sci Res 2012; 8:207–214.
Snedecor WG, Cochran GW. Statistical methods. 10th ed. USA: Lowa State University Press 1982.
Corner EJH. Wayside trees of Malaya. Malayan Nature Society, 1997. 4th ed. vol. 1.
Benthall AP. Trees of Calcutta and its neighborhood. Calcutta: Thacker Spink and Co; 1946.
Clark WC, Thaman RR. Agroforestry in the Pacific islands: systems for sustainability. Tokyo: United Nations University Press; 1993.
Mate GS, Naikwade NS, Chowki AA, Patil SB. Evaluation of anti-nociceptive activity of Cissus quadrangularis on albino mice. Int J Green Pharm 2008; 2:118–121.
Charpentier J. Analysis and measurement of pain in animals. A new conception of pain. In: Soulairac A, Cahn J, Charpentier J, editors. Pain. London, UK Academic Press. 1968. pp. 171–200.
Shah BN, Seth AK. Medicinal plants as a source of anti-pyretic agents a review. Adv Biores 2010; 1:10–16.
Aman AA, Patrick NO. Investigation of anti-inflammatory, antipyretic and analgesic effect of Yemeni Sid honey. World Acad Sci Eng Technol 2011; 80:47–52.
Cheng L, Ming-liang H, Lars B. Is COX-2 a perpetrator or a protector? selective COX-2 inhibitors remain controversial. Acta Pharmacol Sin 2005; 26:926–933.
26.Vane JR, Botting RM. New insight into the mode of action of anti-inflammatory drugs. Inflamm Res 1995; 44:1–10.
Zurier B. Essential fatty acid and inflammation. Ann Rheum Dis 1991; 50:745–746.
Lim JC, Park JH, Budesinsky M, Kasal A, Han YH, Koo BS et al.
Antimutagenic constituents from the thorns of Gleditsia sinensis
. Chem Pharm Bull (Tokyo) 2005; 53:561–564.
Simopoulos AP. Review: omega-3 fatty acids in inflammation and autoimmune diseases. J Am Coll Nutr 2002; 21:495–505.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]