Antibacterial activities of rhubarb extract and the bioactive compounds against Salmonella
Salmonella is one of the primary causes of food borne illnesses worldwide. In this study, antibacterial properties of rhubarb against
Salmonella were investigated. Initial screening showed that rhubarb root ethanol extract strongly inhibited the growth of Salmonella
serotype typhimurium, and the chloroform fraction was found to be the most active fraction. Five major Anthrsaquinone derivatives
were identifi ed from the chloroform fraction by UPLC-MS/MS, namely emodin, aloe-emodin, rhein, physcion and chysophanol. Of
these fi ve compounds, rhein showed the greatest antibacterial activities against S. typhimurium. Time kill curve assay suggested that
rhein killed the bacteria in a relatively fast rate. Further investigations on the mechanisms revealed that rhein signifi cantly altered
the integrity of the cell membrane, resulting in the loss of barrier function and leakage of the nucleotide. The morphological changes
of S. typhimurium treated with rhein were also observed by scanning electron micrographs.
Anthrsaquinone, Antibacterial, Rhein, Rhubarb, Salmonella
Salmonella is one of the primary causes of food borne diseases
worldwide. In recent years, it was responsible for several worst
food borne illness outbreak in the U.S. history, affecting millions
of people. The United States Center for Disease Control
and Prevention (CDC) estimated that approximately 1.4 million
cases/year in US with ~40,000 confi rmed cases and 1,000 deaths
in the US alone (http://www.cdc.gov/foodsafety/outbreaks). Salmonella
bacteria are zoonotic in nature, not only do they impede
the food quality severely, they are also hazardous to human society
. Salmonellosis is an infection caused by the Salmonella
bacteria. It is characterized by diarrhea, fever and cramps, and
the symptoms usually last four to seven days. Severe illness and
death may occur among very young, old and immunocompromised
patients . Various foods have been involved in the
outbreaks of salmonellosis, including meat products , dairy
foods , and vegetables . Large outbreaks may also associate
with un-pasteurized juice or raw fruits. Half the confi rmed
cases were due to Salmonella serotype typhimurium and Salmonella
One key strategy to reduce food borne illnesses is to prevent
growth of spoilage and pathogenic microorganisms in foods.
A number of synthetic chemical preservatives were developed
for this purpose. However, with the increasing consumer awareness
and concern regarding synthetic chemical additives, foods
preserved with natural additives have become popular in recent
years. But the studies on natural antibacterial agents, especially
their mechanisms are still limited. There is a continuing interest
to search for the new antibacterial compounds, especially those
from medicinal/edible plants [22-23]several medicinal plants
have been shown to possess antibacterial potentials against Salmonella
. Rhubarb is an edible medical plant. Its fresh stems
and petioles are consumed as vegetable and its roots and stems
are used for medicinal purposes. Rhubarb root is one of the best-known traditional Chinese herbal medicines. It was traditionally
used as a laxative, to treat constipation, jaundice, gastro-intestinal
hemorrhage, and ulcers . Modern studies have
revealed its diverse biological activities, including antibacterial
, anticancer , anti-infl ammatory  and antioxidant 
effects. Evidence accumulated in the past showed that rhubarb
and its bioactive components had strong antimicrobial activities
against a number of pathogenic microorganisms, such as Bacteroidesfragilis,
Staphylococcus aureus , Escherichia
coli , Bifi dobacterium adolescentis , Candida albicans,
Cryptococcus neoformans and Trichophyton mentagrophytes
To our knowledge, there is only one recent published study focusing
on the antibacterial activity of rhubarb extract against
S. enteritidis . But only crude extracts was screened in that
study. No bioactive compounds were identifi ed and no mechanisms
were explored. The aim of this study was to assess the
antibacterial activity of rhubarb root against S. typhimurium,
and to further identify the bioactive components. The possible
mechanisms of action of the major bioactive component were
also investigated if such compounds were discovered.
Materials and Methods
A. Plant material, chemicals and reagents.
The root of rhubarb (Rheum palmatum L.) was purchased from
a local market in Shanghai, China. Dimethyl sulfoxide (DMSO),
petroleum ether, chloroform, ethyl acetate, n-butanol, glutaraldehyde
and isoamyl acetate were purchased from Sinopharm
Chemical Reagent Corporation (Shanghai, China). Tryptone
Soy Agar (TSA), Trypticase Soy Broth (TSB) was purchased
from Hangzhou Tianhe Microorganism Reagent Corporation
(Zhejiang, China). Standards of emodin, aloe-emodin, rhein,
chrsysophanol and physcion were purchased from Chengdu Must Biotechnology Corporation (Sichuan, China).
B. Microbial strains.
Salmonella typhimurium CMCC 50041 were purchased from Institute
of Microbiology. Chinese Academy of Science (Beijing, China). The bacteria were cultivated at 37 °C on Tryptone Soy
Agar (TSA) and Trypticase Soy Broth (TSB) mediums.
C. Extraction and fractionation of rhubarb
The dried rhubarb were ground to coarse powder using a grinder
(Jin Sui, JSP-1000A). 100g of powder was extracted thrsee times
with 500 mL absolute ethanol under refl ux for 4 hrs. The extract
solution was separated from residue by fi ltration. The ethanol
extract was then concentrated in a rotary evaporator under vacuum
to obtain the rhubarb crude extract (ECE). For fractionation,
10g of ECE was dispersed in distilled water, followed by
extracting with petroleum ether (PEF), chloroform (CF), ethyl
acetate (EAF) and n-butanol (BF), successively. The solvent of
these four fractions was removed in a rotary evaporator under
vacuum to yield gel like concentrates. The concentrates were
further dried under N2. All dried extracts were stored at -20 °C
D. Disc diffusion assay.
The disc diffusion assay was performed according to a published
method (V. K. Bajpai, Al-Reza, Choi, Lee, & Kang, 2009) with
modifi cations. In brief, 50 µL of S. typhimurium was injected
into 5 mL TSB, and cultured under condition of 37 °C, 150 r/
min, for 6 hrs in a bed temperature incubator. The inoculum
was adjusted with 0.1 M PBS (pH 7.2) to 10.6 CFU/mL. 1 mL
prepared suspension was streaked onto the surface of TSA with
a SS-Spreader, then the inoculum on the plate was allowed to dry
for 10 min in drying oven at 37 °C. 6 mm diameter sterile paper
discs were placed on the surface of agar culture. Afterwards, 5
µL of sample was injected onto the disc. The plates were then
cultured under 37 °C for 22 hrs in a temperature incubator (37
°C). Finally, the diameters of inhibition zones against the tested
bacteria of each paper disc were measured. DMSO was used as
negative control. Tests were performed in triplicate.
E. UPLC-MS/MS Analysis :
Dried CF (1 mg) was reconstituted in 10 mL methanol to make
a sample concentration of 100 µg/mL. The sample solution was
sonicated in an ultrasonic bath at room temperature for 5 min,
and was fi ltered with a 0.22 µm syringe fi lter for UPLC-MS/MS
UPLC was performed using a Waters ACQUITY UPLC™
system, equipped with a binary solvent delivery system, an
autosampler, a thermostat column compartment and a diode array
detector (DAD). A Waters UPLC BEH C18 column, at a column
temperature of 40 °C, was used for separation. The mobile phase
consisted of 0.05% acetic acid in water (A) and acetonitrile (B)
using a gradient program of 30~60% (B) in 0~4.5 min, 60~80%
(B) in 4.5~5.0 min, 80~30% (B) in 5.0~5.1 min, 30% (B) in
5.1~7.0 min. The fl ow rate was 0.4 mL/min. The detection wave
length was set at 268 nm and the UV spectrum was recorded
from 190 to 400 nm.
The mass spectrometric analysis was performed in a Waters
Q-TOF Micro TM mass spectrometer (Milford, MA, USA)
connected to the UPLC via ESI interface. Nitrogen was used
as desolvation gas and ultra-high pure helium was used as the
collision gas. The optimized parameters in the negative ion
mode were as follows: the rate of nitrogen (N2), 800 L/hrs;
desolvation temperature, 450 �; capillary voltage, 2.5 kV; cone
voltage, 35 V; cone gas fl ow, 50 L/hrs. The full-scan MS data
were recorded in the range of m/z 100~1000. A data-dependent
program was used in the UPLC-MS/MS analysis, so that the
protonated or deprotonated ions in MS spectra could be selected
for further MS/MS analysis.
F. Minimum inhibitory and minimum bactericidal concentrations :
The antibacterial activities of the fi ve compounds identifi ed
from CF were further evaluated by measuring their minimum
inhibitory (MIC) and minimum bactericidal concentrations
(MBC). The measurement was followed a NCCLS 96-well plate
micro dilution broth method (NCCLS, 2008) using the plates
purchased from Chengdu Must Biotechnology Corporation
(Chengdu, China). The populations of S. typhimurium were
adjusted to 10.5 CFU/mL. The sample was dissolved in DMSO
and merged into TSB culture at a concentration of 2000 μg/mL.
Serial dilution was then conducted to obtain concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.62 and 7.81 μg/mL. 50 µL
inoculum of tested bacteria was added to each well. The negative
control containing only bacteria suspension. The bacteria were
incubated in 96-well plate at 37 ºC for 24 hrs, covered with a
parafi lm paper. Afterwards, 10 μL of INT (Iodonitrotetrazolium)
was added to each well. Half an hour later, the color changes
were observed with the naked eye, color changes from colorless
to purple were noted as positive. The MIC was defi ned as the
lowest concentration that color change from purple to colorless
occurred. To measure MBC, 50 µL of each well which no color
change occurred, the mixture of samples and the strain was
isolated on sterile TSA poured in Petri dishes, then cultured at 37
ºC for 24 hrs. The MBC was defi ned as the lowest concentration
of sample which no viable bacteria occurred on the agar culture
surface. All analysis was carried out thrsee times.
G. The time kill curve assay.
The time kill curve assay was conducted according to a recent
paper (Vivek K. Bajpai, Sharma, & Baek, 2013). Briefl y,1 mL
bacteria solution of S. typhimurium were inoculated with 4 mL
of TSB broth. Then cultured in 37 ºC for 4 hrs.The bacterial
suspension was centrifuged at 8000 rpm for 10 min and the
supernate was discarded. The precipitate bacterium was resuspended
with 1 mL 0.1 M PBS. Each tube that was used for
the kill-time curve assay contained the re-suspended bacteria
suspension (107 CFU/mL) S. typhimurium in the TSB medium.
The tubes were inoculated with rhein at a concentration of MIC
in 5 mL TSB medium, and cultured at 37 ºC with shaking. The
number of viable cells was detected as followed: 100 µL sample
of each treatment tube was diluted with 0.1M PBS, 10-fold
serial dilutions. Then spread on the surface of TSA. The plates
cultured for 24 hrs at 37 ºC and then counted the colonies. The
controls were inoculated without rhein and each test strain was
tested similarly as mentioned above. Each assay was carried out
H. Nucleotide leakage.
The experiment was implemented according to a published
method (Lou, Wang, Zhu, Ma, & Wang, 2011) with minor
modifi cation. Exponential phase S. typhimurium were washed
with 0.1 M PBS, then re-suspended in PBS. Bacteria were
incubated with rhein at the concentration of 2× MICs, cultured
with shaking at 37 ºC. At the time intervals of 0, 2, 4, 6 and 8 hrs,
strains incubated with 0.1 M PBS without rhein were used as
control. Samples with different time treatment were centrifuged
at 4000 rpm for 10 min and then the supernatant was collected.
The OD260 of the supernate was measured by Pharma Spec
UV-3600 (Shimadzu, Kyoto, Japan) at room temperature. The
controls were tested without adding rhein.
I. Scanning electron microscopic analysis.
To further confi rm the effect of rhein affecting the morphology
of S. typhimurium, a scanning electron microscopic (SEM) assay
was performed according to the method published by Baipai et al
(Vivek K. Bajpai, et al., 2013). Logarithmic phase S. typhimurium
were inoculated with rhein at 2× MICs in TSB medium for 12 hrs
at 37 ºC with shaking. Strains incubated with TSB without rhein
were used as control. The samples were centrifuged at 7500 rpm
for 5 min, and the supernate was removed. Bacteria precipitate were washed with 0.1 M PBS for 3 times, then fi xed with 2.5%
glutaraldehyde for 6 hrs, followed by fi xing with 1% osmic acid
solution for 6 hrs. The samples were dehydrated for 15 mins
with ethanol of different concentrations for as followed: 30 %,
50%, 70%, 85%, 95% and 100%. Then the ethanol was replaced
by isoamyl acetate. The samples were dried with carbon dioxide
(CO2). Lastly, the samples were sputter coated with gold for 2 min, then were observed with scanning electron microscopic (S-
4800; Hitachi, Hitachi City, Japan).
J. Statistical analysis.
One-way analysis of variance (ANOVA) and Duncan’s multiple
range tests were performed to determine signifi cant differences
(p < 0.05) between the means by Statistical Product and Service
Solutions (SPSS v.19.0, IBM, Armonk, NY).
Antibacterial activity of fractions of rhubarb extract.
Antibacterial activities of the crude ethanol extract as well as the fi ve fractions against S. typhimurium were measured by the disc
Diameters of inhibition zone of rhubarb ethanol crude extract (10 mg/mL) and fi ve fractions (10 mg/mL) (A); and of crude
extract (10 mg/mL), chloroform fraction and fi ve compounds (concentrations used in assay were the concentrations of their relevant
concentrations in 10 mg/mL CF) (B). (ECE: the rhubarb ethanol crude extract, PEF: petroleum ether; CF: chloroform; EAF: ethyl
acetate; BF: n-butanol)
The fi ve fractions showed different antibacterial activities with the order CF >PEF >EAF>BF = WF. CF appeared to be the most
effective fractions among all fractions, with diameters of inhibition zones 15.4 ± 0.40 mm.
They were aloe-emodin, rhein, emodin, chrsysophanol and physcion, all of which are Anthrsaquinone derivatives.
UPLC-MS/MS analysis of CF
.CF was analyzed by UPLC-MS/MS
UPLC chrsomatogram of the fi ve major compounds identifi ed from chloroform fraction of rhubarb crude extract (DAD
at 268 nm). 1. Aole-emodin; 2. Rhein; 3.Emodin; 4.Chrsysophanol; 5.Physcion
Five major components were identifi ed by comparing their retention time and MS data with the standards.
Chemical composition of chloroform extraction of rhubarb
They were aloe-emodin, rhein, emodin, chrsysophanol and physcion, all of which are Anthrsaquinone derivatives.
Antibacterial activity of compounds identifi ed from rhubarb :
Antibacterial activities of the fi ve compounds identifi ed from
CF of the rhubarb crude extract were tested again by the disc
diffusion assay. The concentrations of the fi ve compounds used
in this assay were their relevant concentrations in CF. ECE and
CF were included for comparative purpose. Rhein showed the
greatest inhibitory effects for S. typhimurium (15.8 ± 0.42 mm),
almost the same as CF.
Minimum inhibitory and minimum bactericide concentration.
Antibacterial effects of the fi ve major Anthrsaquinone
compounds identifi ed from CF were further checked by measuring their minimum inhibitory concentration (MIC) and
minimum bactericide concentration (MBC) Rhein showed the lowest MIC (250 µg/mL) and MBC values
(500 µg/mL) comparing to the other four compounds. The values
of MIC and MBC of emodin were two times higher than
that of rhein, while the MIC and MBC values of aloe-emodin,
chrsysophanol and physcion were all greater than 1000 µg/mL.
The MIC and MBC of fi ve components from chloroform extraction against S. typhimurium
The time kill curve assay :
The effect of rhein on the number of viable cells of S. typhimurium
were evaluated by the time kill curve assay. After being treated with rhein at 2× MIC or without rhein,
bacterial cells were counted every hour in a course of 5 hrs. The
viable counts with rhein treatment showed a constant reduction
for S. typhimurium. After 5 hrs, the viable counts with rhein
treatment were almost zero, indicating complete inhibition
against the two bacteria.
Effect of rhein on the viability of S. typhimurium (B) from the time kill curve assay.
The optical density at 260 nm of S. typhimurium treated with
rhein increased with a period of 8 hrs comparing to that of the
control. The fi rst two hour saw the sharpest increase, over a
6-fold increase of the UV absorption comparing to the control.
Total nucleotide leakage measured by UV absorption at 260 nm from S. typhimurium (B) treated with rhein.
Scanning electron microscopy.
The Scanning electron microscopy (SEM) was utilized to check
the cell morphology of S. typhimurium with and without treatment
of rhein. Pictures taken from electron micrographs showed
that non-treated cells had no changes in cell morphology, displaying
a regular, intact and smooth surface. But the membrane
of S. typhimurium cells treated with rhein showed obvious rapture.
Scanning electron micrographs of S. typhimurium treated with rhein for 12 hours (A. S. typhimurium treated with control; B. S. typhimurium treated with rhein)
The antibacterial properties of rhubarb have been known for a
long time. Rhubarb extracts and compounds showed inhibitory
effects against a number of microorganisms including both
Gram-negative and Gram-positive bacteria. Nonetheless, very
few attentions have been paid on its antibacterial activities against
Salmonella. Therefore, a systematic approach was adopted in
this study to examine the antibacterial effects of rhubarb against
Salmonella, to identify the major bioactive compound(s) and to
investigate the possible mechanisms. As the fi rst step, rhubarb
crude extract ECE and the fi ve fractions made from ECE were
screened by using disc diffusion assay against S. typhimurium.
There are many different assays for screening antimicrobial
activity. Disc diffusion assays was chosen because it is the most
widely used method for screening antibacterial properties of
natural extracts and compounds. The screening results showed,
for the fi rst time, that rhubarb ECE did signifi cantly inhibit
the growth of S. typhimurium. Among the fi ve fractions from ECE,CF was found to be the most effective, thus contains the
major antimicrobial compounds.
In order to search for major bioactive compounds, CF were
analyzed by UPLC-MS/MS and fi ve major Anthrsaquinone
derivatives were identify. About 200 phytochemicals have been
identifi ed thus far from eighteen species of the genus Rheum
L. They belong to several different groups of compounds
including Anthrsaquinone, anthrsone, stilbene, fl avonoids, acyl
glucoside, and pyrone. Anthrsaquinones have been reported
to be the major antibacterial compounds from several species
of Rheum L. Different Anthrsaquinones, due to their different
chemical structure, appeared to have different antibacterial
activities against different bacteria. So our next step was to look
for the most effective compound(s) that specifi cally inhibited the
growth of S. typhimurium. By conducting disc diffusion assays
again on the fi ve compounds, rhein was found to be the most
effective antibacterial compounds. The effectiveness of rhein
was further confi rmed by the measurement of MIC and MBC
values. Taking together, it is reasonable to believe that rhein is a
major bioactive antibacterial compounds in rhubarb root against
Despite many years of antibacterial studies on rhubarb, the
mechanism of action, especially those associated with the
specifi c bioactive compounds, are still largely unknown. In this
study, we explored the possible mechanisms of antibacterial
activities of rhein against S. typhimurium. Firstly, the time kill
curve assay was performed to determine the rate of the bacteria
being killed by rhein. After being treated with 2× MIC rhein
for 5 hrs, almost no live bacteria can be visualized. Without
rhein treatment, the number of bacteria actually increased. To
understand why and how rhein kill S. typhimurium, the possible
effects of rhein in altering the integrity of cell membrane and
changing the cell morphology were examined. When the
membrane integrity of bacteria is destroyed, cell constituents
would leach out, including small ions like K+, large molecules
like protein, nucleotide. Since nucleotide including DNA and
RNA showed strong UV absorption at 260 nm, the absorbance
at 260 nm has been used as detection index of membrane
integrity. Our results showed signifi cant increase of UV 260
nm absorption with S. typhimurium treated with rhein, clearly
indicated that rhein induced damage to the cell membranes,
which further led to signifi cant leakage of DNA and/or RNA.
The effects of rhein on the morphological and physical changes
of S. typhimurium were checked by SEM. The membrane of S.
typhimurium cells treated with rhein showed obvious rupture.
This change resulted in cell decomposition and death eventually.
A similar study of morphological changes was observed for
Aeromonashydrophila when treated with emodin. How these
Anthrsaquinone compounds alter integrity of cell membrane
remains an open question. Further research is warranted to
fully understand the mechanisms of rhubarb and its bioactive
compounds against Salmonella and other food-borne pathogens.
In conclusion, the results of this study showed that rhubarb root
possessed strong antibacterial activity against Salmonella. Rhein,
an Anthrsaquinones component identifi ed from CF of rhubarb
crude extract, was found to be the major bioactive compound.
It killed Salmonella at a relatively fast rate. Investigation on
the possible mechanism of action suggested that rhein could damage the integrality of cell membrane leading to nucleotide
leakage, and changing the cell morphologies. Further research is
warranted to fully understand the mechanisms in the molecular levels.
- Agarwal SK, Singh SS, Verma S, & Kumar S (2000)
Antifungal activity of Anthrsaquinone derivatives from Rheum
emodi. J Ethnopharmacol 72: 43-46.
- Babu K S, Srinivas P V, Praveen B, Kishore K S, Murty U S,
& Rao J M (2003) Antimicrobial constituents from the rhizomes
of Rheum emodi. Phytochemistry 62: 203-207.
- Bajpai V K, Al-Reza S M, Choi U K, Lee J H, & Kang S
C (2009) Chemical composition, antibacterial and antioxidant
activities of leaf essential oil and extracts of Metasequioa
glyptostroboides Miki ex Hu. Food Chem Toxicol 47: 1876-
- Bajpai V K, Baek KH, & Kang S C (2012) Control of
Salmonella in foods by using essential oils: A review. Food
Research International 45: 722-734.
- Bajpai V K, Sharma A, & Baek KH (2013) Antibacterial mode
of action of Cudrania tricuspidata fruit essential oil, affecting
membrane permeability and surface characteristics of foodborne
pathogens. Food Control 32: 582-590.
- Chen C H, Cheng WF, Su H L, & Lai W S (1962) Studies
on Chinese rhubarb. I. Preliminary study on the antibacterial
activity of Anthrsaquinone derivatives of Chinese rhubarb(Rheum palmatum L.). Yao Xue Xue Bao 13: 757-762.
- Chen CH, Liu MZ, Su HL, Wang CY, & Li DD (1964)
Studies on the Chinese rhubarb. V. Antibacterial properties and
stability of antraquinone derivatives and the antagonism of some
compounds to their inhibitory action. Yao Xue Xue Bao 11: 258-265.
- Choi R J, Ngoc T M, Bae K, Cho HJ, Kim DD, Chun J,
Khan S, & KimY S (2013). Anti-infl ammatory properties of
Anthrsaquinones and their relationship with the regulation of
P-glycoprotein function and expression. Eur J Pharm Sci 48:
- Cyong J, Matsumoto T, Arakawa K, Kiyohara H, Yamada H,
& Otsuka Y (1987) Anti-Bacteroides fragilis substance from
rhubarb. J Ethnopharmacol 19: 279-283.
- el-Gazzar FE, & Marth E H (1992) Salmonellae,
salmonellosis, and dairy foods: A review. J Dairy Sci 75; 2327-
- Golberg D, Kroupitski Y, Belausov E, Pinto R, & Sela, S
(2011) Salmonella Typhimurium internalization is variable in
leafy vegetables and fresh herbs. Int J Food Microbiol 145: 250-
- Han C, Liu G, Li Y, Huang Q, & Wang J (2014) Antimicrobial
effects of cinnamon and rhubarb extracts. Applied Mechanics
and Materials 469: 121-125.
- Han C, Wang J, Li Y, & Cui Y (2013) In vitro antimicrobial
activity and effect on E. coli integrity of cinnamon essential oil
and rhubarb ethanol extract. Food Sci Technol Res 19: 1155-
- Huang Q, Lu G, Shen HM, Chung MC, & OngCN (2007).
Anti-cancer properties of Anthrsaquinones from rhubarb. Med
Res Rev 27: 609-630.
- Humphrsey T, & Jorgensen F (2006) Pathogens on meat and infection in animals - Establishing a relationship using
campylobacter and salmonella as examples. Meat Sci 74: 89-97.
- Kim TG, Kang SY, Jung KK, Kang JH, Lee E, Han HM,
& Kim SH (2001) Antiviral activities of extracts isolated from
Terminalis chebula Retz., Sanguisorba offi cinalis L., Rubus
coreanus Miq. and Rheum palmatum L. against hepatitis B
virus. Phytother Res 15: 718-720.
- Lee M H, Kwon HA, Kwon DY, Park H, Sohn DH, Kim
YC, Eo SK, Kang HY, Kim SW, & Lee JH (2006) Antibacterial
activity of medicinal herb extracts against Salmonella. Int J
- Lou Z, Wang H, Zhu S, Ma C, & Wang Z (2011) Antibacterial
activity and mechanism of action of chlorogenic acid. J Food Sci
- Lu C, Wang H, Lv W, Xu P, Zhu J, Xie J, Liu B, & Lou
Z (2011) Antibacterial properties of Anthrsaquinones extracted
from rhubarb against Aeromonas hydrophila. Fisheries Science
- NCCLS (2008) Performance Standards for Antimicrobial
Susceptibility Testing; Ninth Informational Supplement.
NCCLS document M100-S9. In (pp. 120–126). Wayne, PA:
National Committee for Clinical Laboratory Standards.
- Öztürk, M., Aydoğmuş-Öztürk, F., Duru, M. E., & Topçu, G.
(2007). Antioxidant activity of stem and root extracts of Rhubarb
(Rheum ribes): An edible medicinal plant. Food Chemistry,
- Saleem M, Nazir M, Ali MS, Hussain H, Lee YS, Riaz N,
& Jabbar A (2010) Antimicrobial natural products: An update
on future antibiotic drug candidates. Nat Prod Rep 27: 238-254.
- Savoia D (2012) Plant-derived antimicrobial compounds:
Alternatives to antibiotics. Future Microbiol 7: 979-990.
- Wang J, Zhao H, Kong W, Jin C, Zhao Y, Qu Y, & Xiao
X (2010) Microcalorimetric assay on the antimicrobial property
of fi ve hydroxyAnthrsaquinone derivatives in rhubarb (Rheum
palmatum L.) to Bifi dobacterium adolescentis. Phytomedicine
- Zhang X R, Wang JB, Xiao XH, Liu TS, Chu XH, Zhou CP,
& Jin C (2010) Antimicrobial activity and chemical differences
between the two chemotypes of rhubarbs. Yao Xue Xue Bao 45:
- Zheng Qx, Wu Hf, Guo J, Nan Hj, Chen Sl, Yang Js, & Xu
Xd (2013) Review of Rhubarbs: Chemistry and Pharmacology.
Chinese Herbal Medicines 5: 9-32.