Original Article

Yonago Acta medica 2016;59:61–65

In Vitro Inhibition of Cytopathic Effect of Influenza Virus and Human Immunodeficiency Virus by Bamboo Leaf Extract Solution and Sodium Copper Chlorophyllin
Akiko Ito,* Akeno Tsuneki,† Yu Yoshida,* Kazuo Ryoke,* Toshiyuki Kaidoh‡ and Seiji Kageyama†*Division of Oral and Maxillofacial Biopathological Surgery, Department of Medicine of Sensory and Motor Organs, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503, Japan, †Division of Virology, Department of Microbiology and Immunology, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503, Japan and ‡Department of Anatomy, Tottori
University Faculty of Medicine, Yonago 683-8503, Japan.

ABSTRACTthe major ingredient of bamboo leaf extract solution byblocking adsorption. This mechanism of action is different completely from the one of povidone-iodine.

Background

Although the link between oral and oropharyngeal health status and susceptibility to infection
has long been recognized, there is a limit to the selectionof antiseptics for oral care.
Methods Madin-Darby canine kidney (MDCK) cells
were exposed to influenza virus and cultured in thepresence or absence of test reagents: bamboo leaf extract solution and sodium copper chrolophyllin. MDCK
cells were pre-incubated with the reagents to assess theinhibitory activity at adsorption (viral attachment). Similarly, anti-HIV activity and the inhibitory mechanism atadsorption were assessed by MT-2 cell culture system.
Mixture of HIV and bamboo leaf extract solution was
fixed and examined by transmission electron microscopy.
Results The 50% inhibitory concentration (IC50) ofbamboo leaf extract solution against influenza virus
and the 50% cytotoxic concentration (CC50) in MDCK
cells of the solution lay between 0.0313–0.0625% and
0.5–1.0%. The solution inhibited the influenza virus
adsorption at the concentration of 0.5% (P < 0.05). The
values of IC50 and CC50 of sodium copper chlorophyllin
lay between 50–100 µM and 200–400 µM, respectively.
This inhibited the virus adsorption at 200 µM (P < 0.05).
The bamboo leaf extract solution showed values of IC50
against HIV and CC50 in MT-2 cells at around 0.0313%
and between 0.25–0.5%, respectively. This solution inhibited HIV adsorption at 1.25% (P < 0.05). The IC50
and CC50 of sodium copper chlorophyllin lay between
50–100 µM and 200–400 µM, respectively. Sodium
copper chlorophyllin inhibited HIV adsorption at 2.5
mM (P < 0.05). HIV particles survived after the exposure to 0.5% bamboo leaf extract solution.
Conclusion Sodium copper chlorophyllin exerted
antiviral activities against influenza virus and HIV as

Key words

adsorption; bamboo leaf extract solution;
HIV; influenza; human; sodium copper chlorophyllin

The accumulation of dental plaque and colonization oforal surfaces with respiratory pathogens serves as a reservoir for recurrent lower respiratory tract infections.1–5
Indeed, professional oral health care by dental hygienists
is effective in preventing respiratory infections in elderly
persons requiring nursing care.6–9 Although the linkage
between oral and oropharyngeal health status and susceptibility to infection has long been recognized, there isa limit to the selection of antiseptics for oral care.10, 11
Povidone-iodine is one such antiseptic and considered to have the broadest spectrum of antimicrobial action compared with other common antiseptics.12, 13 Oxidative potency of povidone-iodine enables destructionof various structures and enzymes of microbes and viruses.14 However, the antiseptic has a limitation leading
to the possible cytotoxic potential to mammalian cells
through this mechanism of pathogen killing.15–17 Therefore, further exploration is required for the developmentof new materials that have different anti-pathogenic
mechanisms.
Influenza viruses cause a viral respiratory infection
particularly among high-risk individuals, such as children,
aged persons, and patients with chronic disorders. Some
reports indicate that the unclean condition in the oral cavity
promotes influenza infection,18 and good oral hygiene minimizes the severity of respiratory illness.3, 19 Human immunodeficiency virus (HIV) infected approximately 34
million individuals causing acquired immune deficiency
syndrome (AIDS) worldwide. One investigation foundthat 76.7% of HIV-infected individuals showed oral
symptoms among tested AIDS cases.20 Among theseHIV-infected individuals, gum bleeding is observed frequently due to corruption of mucous membrane of theoral cavity after the progress of caries.21–23 Such bleeding from the oral cavity may increase the possibility of
HIV transmission to partners. Therefore, it is indispens-

Corresponding author: Seiji Kageyama, MD, PhD
skageyama@med.tottori-u.ac.jp
Received 2015 December 7
Accepted 2015 December 18
Abbreviations: CC50, 50% cytotoxic concentration; D-MEM, Dulbecco’s modified eagle medium; HIV, Human immunodeficiency
virus; IC50, 50% inhibitory concentration; MDCK, Madin-Darby
canine kidney; TCID50, Median tissue culture infectious dose61

A. Ito et al.

able for such individuals to efficiently improve their own
oral care with potent antimicrobial activity for prevention of infection and disease development.
In the present study, antiviral activities of a plant extract and its ingredient against influenza virus and HIV
were assessed for antiseptic use.

fetal bovine serum, 200 units/mL penicillin G, and 100µg/mL streptomycin at 37 ℃ under a 5% CO2/95% air
atmosphere for four days. Control cells were not exposedto the virus. The total viable cells were counted by trypan blue dye exclusion method on day 4.
MT-2 cells were pre-incubated to the bamboo leaf
extract solution (up to 10%), sodium copper chlorophyllin (up to 5 mM) or test reagent-free culture medium for
10 min to assess the inhibitory activity at adsorption.
The pre-incubated cells were washed extensively to beable to eliminate antiviral activity of test reagents remaining in the culture medium, and then they were exposed to HIV-1LAI (1.3x10-4 TCID50 per cell). In addition
to these pre-incubated cells, control wells were preparedto evaluate growth of the cells in a virus-free condition.
Then, virus-exposed and mock-exposed cells were cultured in the absence of test reagents for three days. The
total viable cells were counted on day 3.

MATERIALS AND METHODS
Influenza virus cytopathic assay

Madin-Darby canine kidney (MDCK) cells were seeded(30,000 per well) in 96-plate and incubated for approximately 24 hours. Then, the semi-confluent cells were
washed twice with phosphate-buffered saline, exposed toinfluenza virus [A/Tottori/ST215/2009(H1N1)] at a doseof 108 copies per well and cultured in the presence or absence of various concentrations of test reagents: bamboo
leaf extract solution (kindly provided by Environmental
Plant Industry, Yonago, Japan), sodium copper chlorophyllin (Wako, Osaka, Japan), and povidone-iodine(‘Isodine gargle’, Meiji, Tokyo, Japan). The culture wasmaintained in Dulbecco’s modified eagle medium (DMEM) supplemented with 10 µg/mL trypsin (Beckton
Dickinson, Tokyo, Japan), 0.2% heat-inactivated bovine
serum albumin, 200 units/mL penicillin G, and 100 µg/
mL streptomycin at 34 ℃ under a 5% CO2/95% air atmosphere for three days. Control cells were not exposedto the virus. The total viable cells were counted by asensitive colorimetric assay (Cell Counting Kit-8, Wako,
Osaka) on day 3.
The semi-confluent MDCK cells were pre-incubatedwith the bamboo leaf extract solution (0.5 and 1%), sodium copper-chlorophyllin (200 and 400 µM) or test
reagent-free culture medium for 10 min to assess theinhibitory activity at adsorption (viral attachment). The
pre-incubated cells were washed extensively to be able toignore antiviral activity of the remaining test reagents inthe culture medium, and then exposed to the influenza
virus (5x107 copies per well). In addition to these preincubated cells, control wells were prepared to evaluate growth of the cells in a virus-free condition. Then,virus-exposed and mock-exposed cells were cultured in
the absence of test reagents for three days. The total viable cells were counted on day 3.

Transmission electron microscopy

The HIV-1LAI (50 µL, 4,000 TCID50 per mL) was subjected to Viro-adembeads (ADEMTEC, Pessac, France)
according to manufacturer’s instruction. The HIV-1LAI
captured with Viro-adembeads was incubated withan equal volume of bamboo leaf extract solution at an
ambient temperature for 5–10 min. The mixture wasthen fixed with 2.5% glutaraldehyde at 4 ℃ for morethan one hour, washed twice with 70% ethanol and resuspended with phosphate buffered saline (100 µL). The
fixed virus particles were postfixed in 1% osmium tetroxide, and stained with 1% uranyl acetate for 1h. They
were then dehydrated in graded ethanol, and embedded
in Epon. Thin sections were stained with uranyl acetate
and lead citrate and examined with a JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan).
Statistical analysis

Statistical analyses were performed using the JMP 9
software (SAS Institute, Cary, NC). The increase in the
total viable cells was evaluated by Dunett’s test, and P <0.05 was regarded as statistically significant.
RESULTS
Anti-influenza virus activity of bamboo leaf extract
solution and sodium copper chlorophyllin and its
inhibitory mechanism

HIV cytopathic assay

MT-2 cells (105 cell per mL) were exposed to of HIV1LAI at 1.3x10-4 median tissue culture infectious dose
(TCID50) per cell, and cultured in the presence or absence of various concentrations of bamboo leaf extract
solution, sodium copper chlorophyllin, and povidoneiodine on day 0. The culture was maintained in RPMI1640 medium supplemented with 10% heat-inactivatedOriginal bamboo leaf extract solution containing 3.3–3.9
mM sodium copper chlorophyllin was defined as 100%concentration in the present experiment. The 50% inhibitory concentration (IC50) of bamboo leaf extract solution lay between 0.0313 and 0.0625%. The 50% cytotoxic concentration (CC50) of the solution lay between 0.5
62Bamboo leaf/Chlorophyllin as antivirals

and 1% (Fig. 1A). The solution exhibited pretreatment
effect and inhibited the influenza virus adsorption at aconcentration of 0.5% (P < 0.05) (Fig. 1B). The valuesof IC50 and CC50 of sodium copper chlorophyllin lay
between 50–100 µM and 200–400 µM, respectively (Fig.
1C). Sodium copper chlorophyllin also exhibited virus
adsorption at 200 µM (P < 0.05) (Fig. 1D).

Anti-influenza virus and anti-HIV activities of povidone-iodine ‘Isodine gargle’

Anti-HIV activity of bamboo leaf extract solution
and sodium copper chlorophyllin and its inhibitory
mechanism

Morphology of HIV particles after exposure tobamboo leaf extract solution

No antiviral activity can be observed against influenza
virus and HIV although the povidone-iodine concentration increased to a level close to cytotoxic concentration:
0.125–0.25% of available iodine in MDCK cell culture(Fig. 3A) and 0.0313–0.0625% in MT-2 cells (Fig. 3B).

Stability of HIV particles was analyzed in the concentration with potent antiviral activity in the HIV culture.
Intact HIV particles could be observed even in the presence of 0.5% bamboo leaf extract solution (Fig. 4). This
concentration is sufficient to exhibit an anti-HIV activity
in culture (0.5%, in Fig. 2A), and corresponds to 16-foldof IC50 and around CC50.

The bamboo leaf extract solution showed values of IC50
and CC50 at around 0.0313% and between 0.25–0.5%,
respectively (Fig. 2A). This solution exhibited pretreatment effect on the inhibition of HIV adsorption at theconcentration of 1.25% (P < 0.05) (Fig. 2B). As with theanti-influenza virus activity, the IC50 and CC50 of sodium copper chlorophyllin lay between 50–100 µM and
200–400 µM, respectively (Fig. 2C). Sodium copper
chlorophyllin also exhibited pretreatment effect and inhibited HIV adsorption at 2.5 mM (P < 0.05) (Fig. 2D).

100,000	  	
  DISCUSSION
Antiviral activity of bamboo leaf extract solution wasrecognized as an agent that fights against the influenza

MDCK	
  cells	
  (per	  well)MDCK	
  cells	
  (per	  well)Fig. 1AA

100,000	
  	
  80,000	  	
  60,000	  	
  40,000	  	
  20,000	
  	
  
0	
  	
  

80,000	  	
  60,000	  	
  40,000	  	
  20,000	
  	
  
0	
  	
  

0	
  

0.0313	
   0.0625	
   0.125	
  

0.25	
  

0.5	
  

1	
  

MDCK	
  cells	
  (per	  well)MDCK	
  cells	
  (per	  well)CC100,000	
  	
  80,000	  	
  60,000	  	
  40,000	  	
  20,000	
  	
  
0	
  	
  

80,000	  	
  60,000	  	
  40,000	  	
  20,000	
  	
  
0	
  	
  

0	
  	
  

50	
  	
  

100	
  	
  

200	
  	
  

400	
  	
  

800	
  	
  

1,600	  	
  Sodium copper chlorophyllin (µM)*

bamboo leaf
extraction (%)

bamboo leaf extraction (%)
100,000	
  	
  

BB

DD

*

Sodium copper
chlorophyllin (µM)

Fig. 1. Inhibition of the cytopathic effect of influenza virus by bamboo leaf extract solution and sodium copper chlorophyllin. A: MDCK
cells were exposed to influenza virus, and cultured in the presence or absence of various concentrations of bamboo leaf extract solution (solid
column). Control cells were not exposed to the virus (open column). Data were expressed as mean ± standard error (n = 4). B: MDCK
cells were pre-incubated to the bamboo leaf extract solution or medium and then exposed to the influenza virus after extensive washing.
Cell culture was carried out in the absence of test reagent. The total viable cells were counted on day 3. C: The viable MDCK cells exposed to influenza virus were cultured in the presence or absence of the sodium copper chlorophyllin. D: Pre-incubated MDCK cells tothe sodium copper chlorophyllin or medium were exposed to the influenza virus after extensive washing. Cell culture was carried out inthe absence of test reagent. *P < 0.05 by Dunnett’s test. MDCK, Madin-Darby canine kidney.

63

15	
  	
  

AA

MT-­‐2	
  cells	  	
  (x105	  per	
  well)MT-­‐2	
  cells	  	
  (x105	  per	
  well)A. Ito et al.

10	
  	
  
5	
  	
  
0	
  	
  

0	
  

0.0313	
   0.0625	
   0.125	
  

0.25	
  

0.5	
  

BB
*

40	
  	
  
30	
  	
  
20	
  	
  
10	
  	
  
0	
  	
  

1	
  

bamboo leaf
extraction (%)

15	
  	
  

CC

MT-­‐2	
  cells	  	
  (x105	  per	
  well)MT-­‐2	
  cells	  	
  (x105	  per	
  well)bamboo leaf extraction (%)

10	
  	
  
5	
  	
  
0	
  	
  

0	
  	
  

50	
  	
  

100	
  	
  

200	
  	
  

400	
  	
  

Fig. 3

DD

40	
  	
  

*

30	
  	
  
20	
  	
  
10	
  	
  
0	
  	
  

800	
  	
   1,600	
  	
  Sodium copper
chlorophyllin (mM)

Sodium copper chlorophyllin (µM)

100,000	
  	
  

A

14	
  	
  

80,000	  	
  MT-­‐2	
  cells	  (x105)

MDCK	
  cells	
  (per	  well)Fig. 2. Inhibition of the cytopathic effect of HIV by bamboo leaf extract solution and sodium copper chlorophyllin. The entire procedure
was carried out similarly to the inhibition assay of cytopathic effect of influenza virus except susceptible cells and viral inoculum: MT-2
cells and HIV. *P < 0.05 by Dunnett’s test. MDCK, Madin-Darby canine kidney; HIV, Human immunodeficiency virus.

60,000	  	
  40,000	  	
  20,000	
  	
  

B

12	
  	
  
10	
  	
  
8	
  	
  
6	
  	
  
4	
  	
  
2	
  	
  
0	
  	
  

0	
  	
  

ConcentraDon	
  (available	
  Iodine	  %)Fig. 3. Inhibition of the cytopathic effect of influenza virus and HIV. A: MDCK cells were exposed to influenza virus and cultured in thepresence or absence of povidone-iodine (solid column). Control cells were not exposed to the virus (open column). The reagent concentration was increased up to the cytotoxic level (0.25%). B: Similarly, MT-2 cells were exposed to HIV and cultured. There was no viable cell
at 0.125% on day 3. MDCK, Madin-Darby canine kidney; HIV, Human immunodeficiency virus.

64

Bamboo leaf/Chlorophyllin as antivirals

as this inhibitory mechanism has been estimated as nonspecific interaction against several different microbes.
29–32
The mechanism of action must be different fromthe one of povidone–iodine.12, 33 The extract solution
and sodium copper chrolophyllin kept antiviral activities
for the entire culture period, but povidone-iodine did notinhibit cytopathic effect even at a subtoxic concentration. Povidone-iodine must exert antiviral activity forshort instances such as one minute incubation, and giveirreversible damage to viral particles as summarized
before.12 This difference was also supported by the factthat HIV particles were not destroyed after 5–10 min exposure to subtoxic concentration of bamboo leaf extract
solution, and could be recognized by electron microscopy. However, additional morphological examination has
to be carried out also in the presence of povidone iodine
for further discussion of the difference between antiviralmechanisms of the bamboo leaf extract and povidone
iodine.
In the current study, sodium copper chlorophyllin
exerted antiviral activities against influenza virus andHIV as the major ingredient of bamboo leaf extract solution by blocking viral-cell interaction (adsorption step)and interfering with the internalization of viruses andresultant replication. This mechanism of action is completely different from the one occurring with povidoneiodine.

HIV

Magnet	  beads
Fig. 4. Intact HIV particles after exposure to higher concentrationof bamboo leaf extract. Typical HIV particles approximately 120
nm in diameter with a conical capsid can be seen even after incubation with 0.5% bamboo leaf extract solution for more than fiveminutes. The diameter of magnet beads ranges from 100–500 nm
according to manufacturer (Ademteck). Bar = 100 nm. MDCK,Madin-Darby canine kidney; HIV, Human immunodeficiency
virus.

Acknowledgments: The authors wish to thank the Environmental
Plant Industry for providing bamboo leaf extract.
The authors declare no conﬂict of interest.

virus and HIV, and was exhibited presumably by inhibition of viral adsorption to cells as reported for othernatural ingredients, such as black tea, coffee, Morinda
citrifolia leaves, manuka honey and bananas.24–28 One
ingredient, sodium copper chrolophyllin, also had antiviral activity against influenza and HIV through the sameantiviral mechanism of the extract solution. The activityof this extract solution may be due to this ingredient.
The IC50 of the extract solution lay between 0.0313 and0.0625% and must contain 116–230 µM sodium copper chlorophyllin. Similarly, the IC50 of the compound,sodium copper chrolophyllin, lay between 50–100 µM.
These two IC50 values of sodium copper chlorophyllin
as ingredients and compound were almost in the same
concentration range. These results suggested sodium
copper chlorophyllin exerted antiviral activities against
influenza and HIV as the major ingredient of bamboo
leaf extract solution.
As described above, the extract solution and sodium
copper chlorophyllin exerted antiviral activities by inhibiting the virus-to-cell interaction on the cell surface,

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