# In Vitro Anticancer MCF-7, Anti-Inflammatory, and Brine Shrimp Lethality Assay (BSLA) and GC-MS Analysis of Whole Plant Butanol Fraction ofRheum ribes (WBFRR)

Authors: Jahangir Khan Achakzai; Muhammad Anwar Panezai; Muhammad Ayub Kakar; Abdul Manan Kakar; Shahabuddin Kakar; Javed Khan; Nazima Yousaf Khan; Inayatullah Khilji; Ajab Khan Tareen
Journal: BioMed Research International
 (2019)

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## Abstract

In this study, GC-MS analysis has shown that whole plant butanol fraction ofrheum ribes (WBFRR) comprises of 21 compounds which exhibited anticancer (MCF-7) activity having IC50 value of 36.01± 0.26. MTT assay (MCF-7), Oxidative Burst assay using chemiluminescence technique, and B-Hatching techniques were the methods used for anticancer MCF-7, anti-inflammatory, and Brine Shrimp Lethality Assay (BSLA). GC-MS was used for structural elucidation. Whole plant methanol extract ofrheum ribes (WMERR), whole plant n-hexane fraction ofrheum ribes (WHFRR), and whole plant aqueous fraction ofrheum ribes (WAFRR) were inactive against anticancer (MCF-7) cell line. Whole plant methanol extract ofrheum ribes (WMERR), whole plant aqueous fraction ofrheum ribes (WAFRR) and whole plant butanol fraction ofrheum ribes (WBFRR) showed anti-inflammatory activity on ROS having IC50 value of 23.2±1.9, 24.2±2.7 and 12.0±0.6. Whole plant butanol fraction ofrheum ribes (WBFRR) showed Brine Shrimp Lethality with LD50 693.302 while whole plant methanol extract ofrheum ribes (WMERR) and whole plant aqueous fraction ofrheum ribes (WAFRR) showed high lethality at highest concentration. This study revealed that whole plant butanol fraction ofrheum ribes (WBFRR) exhibited significant anticancer (MCF-7) activity. In the near future, the constituent of whole plant butanol fraction ofrheum ribes (WBFRR) can be the alternative drug against MCF-7 cell line with least toxicity and side effects.


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## Body

## 1. Introduction

Rheum ribes, the subtropical and temperate regional plant, belongs to Polygonaceae family. The English name of this plant is rhubarb which is of three types, for instance, the Rhapontic rhubarb, the Himalayan or Indian rhubarb, and the Chinese rhubarb [1]. Plenty of active phytochemical and crude drugs are present in this plant of Asian countries [2]. The biologically active phytochemicals are flavonoids (quercetin 3-O-rutinoside, quercetin 3-O-rhamnoside, quercetin 3-O-galactoside, and catechin), anthocyanins (cyaniding 3-glucoside and cyaniding 3-rutinoside), stilbene (desoxyrhapontigenin and trans-rhapontigenin), and anthraquinones (emodin, physcion, aloe-emodin, chrysophanol, and rhein). Ethnomedicinal properties ofrheum ribes are laxative, antioxidative, analgestic, antidiabetic, antimutagenic, ascathartic, anti-inflammatory, anticancer, antibacterial, hepatoprotective, antiplatelet, diarrhea, measles, cholagogue, stomachic, smallpox, hemorrhoids, antiemetic, and antipsoriatic [3–10]. In this research study, anticancer MCF-7, anti-inflammatory, and Brine Shrimp Lethality Assay (BSLA) and GC-MS analysis are analyzed.


## 2. Materials and Methods

### 2.1. Plant Material

### 2.2. Extraction

The plantrheum ribes was collected from the mountains of Chaman, Balochistan, Pakistan, and identified by a taxonomist Prof. Dr. Rasool Bakhsh Tareen, Department of Botany, University of Balochistan, Quetta, Pakistan. 3kg whole plant ofrheum ribes was dried in shade and then grinded, soaked in 5 liter of methanol, kept for 7 days, and shaken daily. After a 7-day period, methanol containing whole plant ofrheum ribes was filtered and vaporized with the help of rotary evaporator. Dried semisolid whole plant methanolic extract ofrheum ribes (WMERR) was 48.6 gm. This crude extract 11.5gm was examined for anticancer MCF-7, anti-inflammatory, and Brine Shrimp Lethality Assay (BSLA) while the remaining extract was fractionated with solvents, for instance, n-hexane, aqueous, and butanol.


### 2.3. Fractionation of Crude Extract

In a separatory funnel, with crude extract two solvents were added such as aqueous and n-hexane. The separatory funnel was shaken thoroughly to create two layers such as aqueous layer and n-hexane layer. Both solvents were separately vaporized with the help of rotary evaporator to form whole plant n-hexane fraction ofrheum ribes (WHFRR) 0.1 gm and whole plant aqueous fractionof rheum ribes (WAFRR) 23.6 gm. Both fractions were examined for anticancer MCF-7, anti-inflammatory, and Brine Shrimp Lethality Assay (BSLA) while whole plant aqueous fraction ofrheum ribes (WAFRR) was further fractionated with a solvent such as butanol [11, 12].


### 2.4. Fractionation of Whole Plant Aqueous Fraction ofrheum ribes (WAFRR)

Whole plant aqueous fraction ofrheum ribes (WAFRR) was fractionated with solvent such as butanol.


### 2.5. Formation of Whole Plant Butanol Fraction ofrheum ribes (WBFRR)

In a separatory funnel, with whole plant aqueous fraction ofrheum ribes (WAFRR), two solvents were added such as butanol and aqueous. The separatory funnel was shaken thoroughly to create two layers such as butanol layer and aqueous layer. Both solvents were separately vaporized with the help of rotary evaporator to form whole plant butanol fraction ofrheum ribes (WBFRR) 2.5gm and whole plant aqueous fraction ofrheum ribes (LAFRR). Whole plant butanol fraction ofrheum ribes (WBFRR) was examined for anticancer MCF-7, anti-inflammatory, and Brine Shrimp Lethality Assay (BSLA) [11, 12].


### 2.6. MTT Assay (MCF-7 Cell Lines)

In this assay, Dulbecco’s Eagle modified medium, containing ten percent fetal bovine serum, was used for the culturing of MCF-7 cell lines which were then kept in five percent CO2 and incubated at 37°C. MCF-7 cells were collected when confluency was developed and 8000 cells per well were plated in 96 well flat. After 24 hours, the extracts and fractions with 50 ug/ml were added and then incubated for 48 hours. After incubation, extracts and fractions were removed. To each well, 0.5mg/ml MTT was added and kept in incubator for 3 hours at 37°C. MTT was reduced into Formazan crystals which was then dissolved in 100ul DMSO and at 570 nm absorbance was taken using micro-plate reader (Spectra Max plus, Molecular Devices, CA, USA). In this assay, doxorubicin was used as a standard drug. The decrease in viable cells or percent inhibition was calculated with the help of following formula:% Inhibition = 100 - (mean of O.D. of test compound - mean of O.D. of negative control)/(mean of O.D. of positive control - mean of O.D. of negative control) x 100)For the calculation of IC50 20 mM stock solution of extracts and fractions was diluted into working solution with 50 uM and then in order to get less than 50 percent inhibition, working solution was further diluted in serial dilutions. With the help of EZ-fit5 software, IC50 was calculated [13].


### 2.7. Anti-Inflammatory Assay

Oxidative Burst Assay Using Chemiluminescence Technique. In this technique, 25 ul diluted whole blood HBSS++ and 25 ul of extracts and fractions were incubated for 15 min at 37°C and then plated in 96 well plates. Control wells contain HBSS++ and cells while blank wells contain HBSS++. 25 ul luminol and 25 ul serum opsonized zymosan were added into each well. In terms of relative light units, the level of ROS was recorded in luminometer. In this assay, Ibuprofen with IC50 11.2 ± 1.9 is used as a standard drug [14].


### 2.8. Brine Shrimp Lethality Assay

B-Hatching Techniques. Scatter 50 mg of brine shrimp eggs in a hatching tray which was already half filled with filtered brine solution. Put it in incubator at 23°C. Take 20 mg of extracts and fraction and dissolve it in 2ml of solvent. Transfer 5, 50, and 500 ul from this solution to 3 vials. Leave them overnight to allow the solvent to evaporate. With the help of Pasteur pipette put 10 larvae per each vial. Add 5ml seawater. Under illumination, for 24 hours, incubate them at 25-27°C. For positive and negative controls, add reference cytotoxic drug along with solvent in other vials. For the determination of LD50, Finney computer program was used [15–17].


### 2.9. Gas Chromatography Mass Spectrometry (GC-MS) Analysis: Triple Quadrupole Acquisition Method MS Parameters

For identification and quantification ofrheum ribescompounds, 2 ul ofrheum ribes extract or fraction was directly injected into the gas chromatograph mod.6890N Network GC System (Agilent Technologies Palo Alto, CA) together in the presence of mass spectrometer mod. 5973 Network Mass Selective Detector (Agilent Technologies Palo Alto, CA) and furnished in the presence of a column HP-5MS (30 m length, 0.25 mm interior diameter, 0.25 um film width Agilent Technologies, Palo Alto, CA). Helium gas was off. Injection was made into a split-splitless injector (split ratio 30:1) at 250°C. The oven program was as follows: 70°C for 3 min, then 6°C/min to 180 for 5 min, then 6°C/min to 280°C for 10 min, then 8°C/min to 290°C for 20 min. The MSD transfer line was set at a temperature of 250°C; MSD temperature quadrupole was of 150°C and ionization temperature was 230°C; mass spectra were seventy electrovolts and scan achievement was accomplished in the series between thirty five and 300m/z. The identification of the components of therheum ribes extract or fraction was assigned by matching their mass spectra with those available in the libraries NIST 02 and WILEY [18].


## 2.1. Plant Material

## 2.2. Extraction

The plantrheum ribes was collected from the mountains of Chaman, Balochistan, Pakistan, and identified by a taxonomist Prof. Dr. Rasool Bakhsh Tareen, Department of Botany, University of Balochistan, Quetta, Pakistan. 3kg whole plant ofrheum ribes was dried in shade and then grinded, soaked in 5 liter of methanol, kept for 7 days, and shaken daily. After a 7-day period, methanol containing whole plant ofrheum ribes was filtered and vaporized with the help of rotary evaporator. Dried semisolid whole plant methanolic extract ofrheum ribes (WMERR) was 48.6 gm. This crude extract 11.5gm was examined for anticancer MCF-7, anti-inflammatory, and Brine Shrimp Lethality Assay (BSLA) while the remaining extract was fractionated with solvents, for instance, n-hexane, aqueous, and butanol.


## 2.3. Fractionation of Crude Extract

In a separatory funnel, with crude extract two solvents were added such as aqueous and n-hexane. The separatory funnel was shaken thoroughly to create two layers such as aqueous layer and n-hexane layer. Both solvents were separately vaporized with the help of rotary evaporator to form whole plant n-hexane fraction ofrheum ribes (WHFRR) 0.1 gm and whole plant aqueous fractionof rheum ribes (WAFRR) 23.6 gm. Both fractions were examined for anticancer MCF-7, anti-inflammatory, and Brine Shrimp Lethality Assay (BSLA) while whole plant aqueous fraction ofrheum ribes (WAFRR) was further fractionated with a solvent such as butanol [11, 12].


## 2.4. Fractionation of Whole Plant Aqueous Fraction ofrheum ribes (WAFRR)

Whole plant aqueous fraction ofrheum ribes (WAFRR) was fractionated with solvent such as butanol.


## 2.5. Formation of Whole Plant Butanol Fraction ofrheum ribes (WBFRR)

In a separatory funnel, with whole plant aqueous fraction ofrheum ribes (WAFRR), two solvents were added such as butanol and aqueous. The separatory funnel was shaken thoroughly to create two layers such as butanol layer and aqueous layer. Both solvents were separately vaporized with the help of rotary evaporator to form whole plant butanol fraction ofrheum ribes (WBFRR) 2.5gm and whole plant aqueous fraction ofrheum ribes (LAFRR). Whole plant butanol fraction ofrheum ribes (WBFRR) was examined for anticancer MCF-7, anti-inflammatory, and Brine Shrimp Lethality Assay (BSLA) [11, 12].


## 2.6. MTT Assay (MCF-7 Cell Lines)

In this assay, Dulbecco’s Eagle modified medium, containing ten percent fetal bovine serum, was used for the culturing of MCF-7 cell lines which were then kept in five percent CO2 and incubated at 37°C. MCF-7 cells were collected when confluency was developed and 8000 cells per well were plated in 96 well flat. After 24 hours, the extracts and fractions with 50 ug/ml were added and then incubated for 48 hours. After incubation, extracts and fractions were removed. To each well, 0.5mg/ml MTT was added and kept in incubator for 3 hours at 37°C. MTT was reduced into Formazan crystals which was then dissolved in 100ul DMSO and at 570 nm absorbance was taken using micro-plate reader (Spectra Max plus, Molecular Devices, CA, USA). In this assay, doxorubicin was used as a standard drug. The decrease in viable cells or percent inhibition was calculated with the help of following formula:% Inhibition = 100 - (mean of O.D. of test compound - mean of O.D. of negative control)/(mean of O.D. of positive control - mean of O.D. of negative control) x 100)For the calculation of IC50 20 mM stock solution of extracts and fractions was diluted into working solution with 50 uM and then in order to get less than 50 percent inhibition, working solution was further diluted in serial dilutions. With the help of EZ-fit5 software, IC50 was calculated [13].


## 2.7. Anti-Inflammatory Assay

Oxidative Burst Assay Using Chemiluminescence Technique. In this technique, 25 ul diluted whole blood HBSS++ and 25 ul of extracts and fractions were incubated for 15 min at 37°C and then plated in 96 well plates. Control wells contain HBSS++ and cells while blank wells contain HBSS++. 25 ul luminol and 25 ul serum opsonized zymosan were added into each well. In terms of relative light units, the level of ROS was recorded in luminometer. In this assay, Ibuprofen with IC50 11.2 ± 1.9 is used as a standard drug [14].


## 2.8. Brine Shrimp Lethality Assay

B-Hatching Techniques. Scatter 50 mg of brine shrimp eggs in a hatching tray which was already half filled with filtered brine solution. Put it in incubator at 23°C. Take 20 mg of extracts and fraction and dissolve it in 2ml of solvent. Transfer 5, 50, and 500 ul from this solution to 3 vials. Leave them overnight to allow the solvent to evaporate. With the help of Pasteur pipette put 10 larvae per each vial. Add 5ml seawater. Under illumination, for 24 hours, incubate them at 25-27°C. For positive and negative controls, add reference cytotoxic drug along with solvent in other vials. For the determination of LD50, Finney computer program was used [15–17].


## 2.9. Gas Chromatography Mass Spectrometry (GC-MS) Analysis: Triple Quadrupole Acquisition Method MS Parameters

For identification and quantification ofrheum ribescompounds, 2 ul ofrheum ribes extract or fraction was directly injected into the gas chromatograph mod.6890N Network GC System (Agilent Technologies Palo Alto, CA) together in the presence of mass spectrometer mod. 5973 Network Mass Selective Detector (Agilent Technologies Palo Alto, CA) and furnished in the presence of a column HP-5MS (30 m length, 0.25 mm interior diameter, 0.25 um film width Agilent Technologies, Palo Alto, CA). Helium gas was off. Injection was made into a split-splitless injector (split ratio 30:1) at 250°C. The oven program was as follows: 70°C for 3 min, then 6°C/min to 180 for 5 min, then 6°C/min to 280°C for 10 min, then 8°C/min to 290°C for 20 min. The MSD transfer line was set at a temperature of 250°C; MSD temperature quadrupole was of 150°C and ionization temperature was 230°C; mass spectra were seventy electrovolts and scan achievement was accomplished in the series between thirty five and 300m/z. The identification of the components of therheum ribes extract or fraction was assigned by matching their mass spectra with those available in the libraries NIST 02 and WILEY [18].


## 3. Results and Discussion

MCF-7 cell line results have shown that whole plant butanol fraction ofrheum ribes (WBFRR) exhibited anticancer activity against MCF-7 cell line with IC50 value of 36.01± 0.26. Whole plant methanol extract ofrheum ribes (WMERR), whole plant n-hexane fraction ofrheum ribes (WHFRR), and whole plant aqueous fraction ofrheum ribes (WAFRR) were inactive against anticancer (MCF-7) cell line. The anticancer activities (MCF-7 cell line) of extract and fractions of whole plant ofrheum ribes are shown in Table 1.Table 1
Anticancer Assay (MCF-7) of extracts and fractions of whole plant ofRheum ribes.
S.No. Extract/Fraction/Std. Drug Conc. (ug/ml) % Inhibition/Stimulation IC50 ± S.D. 1 WMERR 50 38.17 Inactive  2 WHFRR 50 22.46 Inactive  3 WAFRR 50 9.90 Inactive  4 WBFRR 50 93.15 36.01± 0.26  5 Doxorubicin 50 73.23 0.80± 0.05Whole plant methanol extract ofrheum ribes (WMERR), whole plant aqueous fraction ofrheum ribes (WAFRR), and whole plant butanol fraction ofrheum ribes (WBFRR) showed anti-inflammatory activity on ROS having IC50 value of 23.2±1.9, 24.2±2.7 and 12.0±0.6. The anti-inflammatory activities of extract and fractions of rheum ribes are shown in Table 2.Table 2
Anti-inflammatory activities of extracts and fractions of whole plant ofRheum ribes.
S.NO Extract/Fraction/Std. Drug Conc. (ug/ml) % Inhibition/Stimulation IC50 ± S.D. 1 WMERR 250,50,10 - 23.2± 1.9  2 WHFRR 250,50,10 - -  3 WAFRR 250,50,10 - 24.2± 2.7  4 WBFRR 250,50,10 - 12.0± 0.6  5 Ibuprofen   11.2± 1.9 ug/mlwhole plant butanol fraction ofrheum ribes (WBFRR) showed Brine shrimp lethality with LD50 693.302 while whole plant methanol extract ofrheum ribes (WMERR) and whole plant aqueous fraction ofrheum ribes (WAFRR) showed high lethality at highest concentration. Brine shrimp lethality assay of extract and fractions of rheum ribes is shown in Tables 3–6.Table 3
Brine Shrimp Lethality Bioassay of Whole plant Methanol Extract ofRheum ribes (WMERR).
Dose (ug/ml) No. of Shrimps No. of Survivors LD50 (ug/ml) STD. Drug LD50 (ug/ml) 10 30 28 - Etoposide 7.4625 100 30 27 1000 30 00Table 4
Brine Shrimp Lethality Bioassay of Whole Plant n-Hexane Fraction ofRheum ribes(WHFRR).
Dose (ug/ml) No. of Shrimps No. of Survivors LD50 (ug/ml) STD. Drug LD50 (ug/ml) 10 30 28 - Etoposide 7.4625 100 30 24 1000 30 00Table 5
Brine Shrimp Lethality Bioassay of Whole Plant Aqueous Fraction ofRheum ribes (WAFRR).
Dose (ug/ml) No. of Shrimps No. of Survivors LD50 (ug/ml) STD. Drug LD50 (ug/ml) 10 30 29 - Etoposide 7.4625 100 30 24 1000 30 00Table 6
Brine Shrimp Lethality Bioassay of Whole Plant Butanol Fraction ofRheum ribes (WBFRR).
Dose (ug/ml) No. of Shrimps No. of Survivors LD50 (ug/ml) STD. Drug LD50 (ug/ml) 10 30 30 693.302 Etoposide 7.4625 100 30 29 1000 30 11Molecular formula, molecular mass, structure, and RT of compounds 1-21 of whole plant butanol fraction ofrheum ribes (WBFRR) are shown in Tables 7–10 while mass spectra interpretation of compounds 1-21 of whole plant butanol fraction ofrheum ribes (LBFRR) is presented in Tables 11–14.Table 7
Molecular formula, molecular mass, structure, and RT of compounds 1-5 whole plant butanol fraction ofrheum ribes (WBFRR).
Compound Name Molecular Formula Molecular Mass Structure RT 1 1H-Inden-1-one, 2,3-dihydro-3,3,6-trimethyl- C12H14O 174  21.411  2 Butanoic acid, 4-(1,1-dimethylethoxy)-3-hydroxy-, methyl ester, (R)- C9H18O4 190  22.644  3 Octaethylene glycol monododecyl ether C28H58O9 538  22.804  4 Silane, (diphenylmethoxy)trimethyl- C16H20OSi 256  23.140  5 Tetradecanoic acid, 2-hydroxy- C14H28O3 244  24.015Table 8
Molecular formula, molecular mass, structure, and RT of compounds 6-10 whole plant butanol fraction ofrheum ribes (WBFRR).
Compound Name Molecular Formula Molecular Mass Structure RT 6 Butanedioic acid, dibutyl ester C12H22O4 230  25.151  7 Butanedioic acid, hydroxy-, dibutyl ester, (±)- C12H22O5 246  26.902  8 Pipradrol C18H21NO 267  27.076  9 Tetradecanoic acid C14H28O2 228  30.280  10 n-Hexadecanoic acid C16H32O2 256  39.870Table 9
Molecular formula, molecular mass, structure, and RT of compounds 11-15 whole plant butanol fraction ofrheum ribes (WBFRR).
Compound Name Molecular Formula Molecular Mass Structure RT 11 Hexanedioic acid, bis(2 methylpropyl) ester C14H26O4 258  40.231  12 Butyl citrate C18H32O7 360  44.843  13 2-Bromotetradecanoic acid C14H27BrO2 307  45.437  14 (2,3-Diphenylcyclopropyl)methyl phenyl sulfoxide, trans- C22H20OS 332  48.124  15 9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol, (3β,5Z,7E)- C27H44O3 416  48.653Table 10
Molecular formula, molecular mass, structure, and RT of compounds 16-21 whole plant butanol fraction ofrheum ribes (WBFRR).
Compound Name Molecular Formula Molecular Mass Structure RT 16 Ethyl iso-allocholate C26H44O5 436  54.708  17 β-D-Mannofuranoside, 2,3:5,6-DI-O-ethylboranediyl-1-O-(stigmasta-5, 22-dien-3-yl)- C39H64B2O6 650  55.215  18 9,19-Cyclolanost-24-en-3-ol, acetate, (3β)- C32H52O2 468  55.985  19 Stigmastan-3,5-diene C29H48 396  56.278  20 Vitamin E C29H50O2 430  56.655  21 9,19-Cyclolanost-24-en-3-ol, acetate, (3β)- C32H52O2 468  57.338Table 11
Mass spectra of compounds 1-5 of whole plant butanol fraction ofrheum ribes (LBFRR).
Compound m/z ( % relative abundance) 1 174(M+], 300), 160(125), 159(999), 131(90), 116(110), 115(172), 91(107), 51(86), 39(116), 27(80)  2 190(M+), 117(238), 104(188), 103(466), 85(191), 71(167), 59(134), 57(999), 43(454), 41(287), 29(219)  3 538(M+), 89(468), 87(229), 73(351), 71(213), 59(182), 57(384), 45(999), 44(232), 43(338), 29(196)  4 256(M+], 378), 241(218), 179(444), 178(240), 168(176), 167(999), 165(400), 152(180), 73(680), 45(244)  5 244(M+), 89(999), 71(389), 69(220), 57(285), 55(454), 43(727), 42(233), 41(610), 29(363), 27(194)Table 12
Mass spectra of compounds 6-10 of whole plant butanol fraction ofrheum ribes (LBFRR).
Compound m/z ( % relative abundance) 6 230(M+), 157(148), 102(45), 101(999), 57(180), 56(159), 55(67), 41(167), 29(133), 28(72), 27(49)  7 246(M+), 173(78), 145(304), 117(170), 90(155), 89(999), 71(214), 57(554), 56(143), 41(262), 29(251)  8 267(M+), 182(16), 165(24), 152(15), 105(69), 85(50), 84(999), 82(12), 77(73), 56(50), 55(12)  9 228(M+), 129(371), 85(241), 73(999), 71(338), 69(282), 60(841), 57(609), 55(451), 43(681), 41(461)  10 256(M+), 129(435), 83(267), 73(980), 71(373), 69(351), 60(999), 57(840), 55(767), 43(817), 41(574)Table 13
Mass spectra of compounds 11-15 of whole plant butanol fraction ofrheum ribes (LBFRR).
Compound m/z ( % relative abundance) 11 258(M+), 185(585), 129(999), 111(264), 101(136), 100(124), 57(463), 56(225), 55(216), 41(216), 29(145)  12 360(M+), 259(159), 186(103), 185(999), 129(676), 111(104), 57(180), 56(86), 43(59), 41(200), 29(118)  13 307(M+), 99(339), 83(299), 73(436), 69(386), 57(616), 55(712), 43(946), 41(999), 29(473), 27(296)  14 332(M+), 207(238), 193(89), 130(103), 129(959), 128(137), 117(83), 115(160), 92(57), 91(999), 77(98)  15 416(M+), 136(999), 118(920), 81(655), 69(672), 67(606), 59(841), 57(713), 55(999), 44(999), 43(999)Table 14
Mass spectra of compounds 16-21 of whole plant butanol fraction ofrheum ribes (LBFRR).
Compound m/z ( % relative abundance) 16 436(M+), 83(460), 81(547), 69(609), 57(797), 55(914), 44(492), 43(999), 41(867), 29(476), 17(469)  17 650(M+), 395(282), 394(440), 255(268), 111(255), 99(245), 83(999), 81(256), 69(441), 57(256), 55(430)  18 468(M+), 121(278), 109(324), 107(311), 95(456), 93(260), 81(316), 69(752), 55(485), 43(999), 41(572)  19 397(M+1], 165), 396(M+], 547), 147(201), 145(157), 105(167), 81(233), 57(269), 55(291), 43(999), 41(209)  20 430(M+], 318), 166(117), 165(999), 164(326), 85(135), 71(226), 57(410), 55(185), 43(419), 41(171)  21 468(M+), 121(278), 109(324), 107(311), 95(456), 93(260), 81(316), 69(752), 55(485), 43(999), 41(572)


## 4. Conclusion

In this study, GC-MS analysis has shown that whole plant butanol fraction ofrheum ribes (WBFRR) is composed of 21 compounds which exhibited anticancer (MCF-7) activity having IC50 value of 36.01± 0.26. Whole plant methanol extract ofrheum ribes (WMERR), whole plant n-hexane fraction ofrheum ribes (WHFRR), and whole plant aqueous fraction ofrheum ribes (WAFRR) were inactive against anticancer (MCF-7) cell line.Whole plant methanol extract ofrheum ribes (WMERR), whole plant aqueous fraction ofrheum ribes (WAFRR), and whole plant butanol fraction ofrheum ribes (WBFRR) showed anti-inflammatory activity on ROS having IC50 value of 23.2±1.9, 24.2±2.7, and 12.0±0.6.Whole plant butanol fraction ofrheum ribes (WBFRR) showed Brine shrimp lethality with LD50 693.302 while whole plant methanol extract ofrheum ribes (WMERR) and whole plant aqueous fraction ofrheum ribes (WAFRR) showed high lethality at highest concentration.This study revealed that whole plant butanol fraction ofrheum ribes (WBFRR) exhibited significant anticancer MCF-7 activity. In the near future, the constituent of whole plant butanol fraction ofrheum ribes (WBFRR) can be the alternative drug against MCF-7 cell line with the least toxicity and side effects.



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*Source: 3264846-2019-06-23.xml*